Therapeutic strategies for the treatment of preeclampsia

ABSTRACT

Compositions and methods for treating preeclampsia in a subject in need thereof are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Application Ser. Nos.61/762,831 and 61/762,830, each filed Feb. 8, 2013, as well as U.S.Application Ser. No. 61/906,074, filed Nov. 13, 2013, each of which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

Hypertension complicates up to 10% of all pregnancies worldwide. In theUnited States, preeclampsia affects 5-7% of all pregnancies,approximately 300,000 pregnancies a year. Yet, it disproportionatelyrepresents 15% of all maternal-fetal morbidity and mortality.Preeclampsia is known to cause immediate maternal-fetal morbidities suchas growth restriction, oligohydramnios, fetal death, maternal seizures,stroke, cerebrovascular hemorrhage, and maternal death (78). Motherswith a history of preeclampsia are at increased risk of future cardiacdisease including myocardial infarction and stroke (24, 55, 56).Children born from preeclamptic pregnancies are also at increased riskof stroke (42), epilepsy (98), and metabolic, nutritional and blooddisease (97) in later childhood or as an adult. Clearly, preeclampsiahas immediate and long term effects on both the fetus and mother.However, its pathogenesis is poorly understood. Consequently,preventative, therapeutic, and curative modalities for preeclampsia areelusive. The only true cure for preeclampsia is the delivery of thefetus and dysfunctional placenta. This delivery is often preterm andcontributes to additional morbidity and mortality (78). This factemphasizes the importance of finding appropriate unifying pathways to beable to treat preeclampsia.

The neurohypophysial hormone, arginine vasopressin (AVP; FIG. 1), is aknown regulator of blood pressure and composition in human and animalmodels. AVP is a major player in blood pressure control in selectedpopulations including African Americans (4), the elderly (21), and inpatients with congestive heart (26) or renal failure (3). This hormoneappears to specifically be causative in patients with low-reninhypertension (81), which makes up a larger portion of the humanessential hypertensive population (27%) than high-renin hypertension(16%) (51). However, whether AVP has a causative role in establishedpreeclampsia has previously been unclear. Establishing such a role forAVP would provide a therapeutic target for the treatment ofpreeclampsia, which has to date remained elusive.

SUMMARY OF THE INVENTION

In a first aspect, a method of treating preeclampsia in a subject inneed thereof, includes administering to the subject a therapeuticallyeffective amount of a pharmaceutical compound that inhibits an argininevasopressin receptor.

In a second aspect, a method of treating preeclampsia in a subject inneed thereof, includes inhibiting production and/or secretion and/oreffects of AVP and/or lowering the concentration of AVP in the blood ofthe subject.

In one embodiment, the method includes administering to the subject atherapeutically effective amount of a pharmaceutical compound thatinhibits production and/or secretion and/or effects of AVP in thesubject.

In another embodiment, the pharmaceutical compound inhibits the effectsof AVP by inhibiting an arginine vasopressin receptor. In a furtherembodiment, the arginine vasopressin receptor includes at least one ofV1A, V2, and V1B.

In one embodiment, the pharmaceutical compound is a vasopressin receptorantagonist. In a further embodiment, the pharmaceutical compound isselected from the group consisting of conivaptan, tolvaptan, andrelcovaptan, and combinations thereof.

In another embodiment, the pharmaceutical compound istetrahydrobiopterin (BH4) or a chemically related compound.

In one embodiment, the amount of the pharmaceutical compound is at leastabout 1 to about 50 mg per day.

In one embodiment, the concentration of AVP is lowered using anextracorporeal therapy technique. In a further embodiment, theextracorporeal therapy technique includes at least one of apheresis,hemodialysis, and hemofiltration.

In a third aspect, a composition for treatment of preeclampsia includesa therapeutically effective amount of a first arginine vasopressinreceptor inhibitor and a therapeutically effective amount of a secondarginine vasopressin receptor inhibitor.

In another embodiment, the first arginine vasopressin receptor inhibitorincludes a first vaptan drug and the second arginine vasopressinreceptor inhibitor comprises a second vaptan drug.

In a fourth aspect, a pharmaceutical dosage form includes atherapeutically effective amount of a first arginine vasopressinreceptor inhibitor, a therapeutically effective amount of a secondarginine vasopressin receptor inhibitor, and one or morepharmaceutically suitable carriers, diluent, and/or excipients.

In one embodiment, the dosage form includes an oral, injection,infusion, inhalation, transdermal, or implant dosage form.

In another embodiment, the pharmaceutical dosage form includes about 1to about 500 mg of the first arginine vasopressin receptor inhibitor andabout 1 to about 500 mg of the second arginine vasopressin receptorinhibitor.

In fifth aspect, a composition for treatment of preeclampsia includes atherapeutically effective amount of tetrahydrobiopterin (BH4) or achemically related compound; and a therapeutically effective amount ofan arginine vasopressin receptor inhibitor.

In a sixth aspect, a method of treating preeclampsia in a subject inneed thereof includes the steps of a) removing whole blood from apatient, b) reducing or removing vasopressin in the removed blood, andc) recirculating the remaining blood components into the bloodstream ofthe patient.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 The protein product of the vasopressin (AVP) gene. The signalsequence targets the protein for cellular export. AVP is then producedand released in a 1:1 molar ratio with the AVP carrier proteinneurophysin II, and with copeptin. While AVP exhibits a very shorthalf-life within the plasma, copeptin is much more stable and isprimarily cleared into the urine where it can be detected easily byimmuno-based assays (5).

FIG. 2. Elevated vasopressin in sRA mice. A: arginine vasopressin (AVP)immunoreactivity in the supraoptic (SON, top and middle rows, from fourseparate animals) and paraventricular (PVN, bottom row, from twoseparate animals) nuclei in female sRA and control animals. Note theincreased numbers of strongly immunoreactive AVP neurons in theretrochiasmatic part of the SON in sRA animals. ON, optic tract; 3V,third ventricle. Bars=200 μm. B: total immunoreactive cell fragments perside, greater than 10 μm in diameter, in four serial sections (spaced200 μm apart) through the PVN and SON of littermate control and sRA mice(n=3 females each group). C: plasma copeptin levels (n=4 male+4 femalecontrol, 4 male+4 female sRA). D: urine copeptin concentration, totaldaily urine volume, and total daily copeptin loss into urine (n=12male+5 female control, 10 male+7 female sRA). All data are means±SE.*P<0.05 vs. control.

FIG. 3. Blood pressure responses to vasopressin receptor antagonists. A:systolic blood pressure (BP), monitored by tail-cuff, at baseline andwith 10 days of chronic subcutaneous infusion (22 ng/h) of the V1/V2nonpeptide antagonist conivaptan (n=2 male+4 female control, 2 male+4female sRA). Hourly telemetric blood pressure (B, MAP) and heart rate(C, HR) recordings for 3 days preceding and 18 days during subcutaneousinfusion of the nonselective V1A/V2 receptor antagonist conivaptan (22ng/h) in a female sRA mouse are shown. D: spontaneous ambulatoryphysical activity counts during conivaptan infusion experiment (in B andC). E: systolic BP, monitored by tail-cuff, at baseline and with 10 daysof chronic subcutaneous infusion (22 ng/h) of the V2-selectiveantagonist tolvaptan (n=4 male+5 female control, 4 male+6 female sRA).F: hourly average radiotelemetric MAP recordings from (n=4 female) sRAmice at baseline and after 10 days of subcutaneous tolvaptan infusion(Drug×Time, P=0.029). All data are means±SE. *P<0.05 vs. control,†P<0.05 vs. baseline sRA.

FIG. 4. Vascular reactivity of abdominal aorta. A: maximum contractileresponse to 100 mmol/1 KCl. B and C: relaxation responses to gradeddoses of acetylcholine and sodium nitroprusside after half-maximalcontraction to PGF2α. D-H: contractile responses to graded doses ofarginine vasopressin, phenylephrine, endothelin-1, angiotensin II, andprostaglandin-F2α (PGF2α) (n=6 male control, 5 male sRA). All data aremeans±SE. *P<0.05 vs. control.

FIG. 5. Mesenteric artery vascular reactivity. A: maximum contractileresponse to 100 mmol/1 KCl. B: contractile responses to graded doses ofarginine vasopressin, phenylephrine, and endothelin-1 (n=6 male control,6 male sRA). C: external and lumen diameters, wall thickness,media-to-lumen ratio, and cross-sectional area of mesenteric arteriesmaintained at 75 mmHg lumen pressure, in calcium-free conditions. D:mesenteric artery mRNA expression of the AVP V1A receptor, theendothelin-1 ETA receptor, RGS2, and RGS5 (V1A, RGS2, and RGS5; n=4male+5 female control, 4 male+3 female sRA. ETA, n=4 male control, 4male sRA). All data are means±SE. *P<0.05 vs. control.

FIG. 6. Serum electrolytes. A: serum sodium concentration. B:serum-ionized calcium concentration (baseline: n=8 male and 12 femalecontrol, 5 male and 8 female sRA; tolvaptan: n=4 male and 5 femalecontrol, 4 male and 6 female sRA). All data are means±SE. *P<0.05 vs.control. † P<0.05 vs. baseline sRA.

FIG. 7. Maternal plasma copeptin, cystatin C, and vasopressinase (LNPEP)protein concentrations by trimester of pregnancy. (A) Compared tonon(pregnant women and women with normotensive pregnancies, plasmacopeptin concentrations were significantly elevated in all threetrimesters of pregnancy in women that eventually developed preeclampsia.Importantly, copeptin was grossly elevated as early as the sixth week ofpregnancy. (B) Plasma cystatin C was affected by gestational age in asimilar manner in women that did or did not experience preeclampsia. (C)Plasma LNPEP was essentially unchanged by gestational age and bypreeclampsia status. *P<0.05 vs. non-pregnant and gestationaltime-matched control pregnant samples.

FIG. 8. Predictive value of maternal plasma copeptin without adjustmentfor any covariates. Receiver operator characteristic (ROC) analyses ofthe utility of copeptin, without correction for covariates, as apredictive tool for the subsequent development of preeclampsia.

FIG. 9. Sufficiency of vasopressin to induce preeclampsia-likephenotypes in C57Bl/6J mice. (A) Vasopressin infusion significantlyreduced fecundity. X2 P<0.005. (B) Vasopressin infusion appears toinduce hypertension and proteinuria in pregnant mice. (C) Images ofexample gestational day 18 fetuses, illustrating substantial fetalgrowth restriction by vasopressin infusion. (D) Electron micrographs ofrenal cortex, illustrating glomerular endotheliosis. Top two panels arefrom a saline infused animal which had a glomerular basement membranethickness within normal limits (thin white arrow). The bottom two panelsare from an animal that received vasopressin infusion. Redundantendothelial cell membrane is present (thick black arrow), and basementmembranes are markedly thickened with electron dense material (thickwhite arrow).

FIG. 10. Chronic vasopressin (AVP) infusion causes a dose- andtime-dependent increase in blood pressure during pregnancy. Chronicsubcutaneous infusion of AVP at 24 ng/hr causes a late-pregnancyincrease in blood pressure that is not achieved by a 10-fold lowerinfusion rate (2.4 ng/hr).

FIG. 11. Effects of 24 ng/hr AVP infusion throughout pregnancy on fetaldevelopment. (A) Photo of uterus and fetuses in situ on GD18 of awildtype mouse chronically infused with AVP. White arrows identify threereabsorbed fetal/placental units within the uterus. (B) Magnified imagesof the six placentas from the pregnant mouse in panel A, illustratingthe necrotic nature and small size of the three placentas identified inpanel A. (C) Photo of uterus and fetuses in situ on GD18 of a secondwildtype mouse chronically infused with AVP. White arrows identify fivereabsorbed fetal/placental units. (D) Magnified images of the fiveplacentas identified in panel C. (E) Magnified images of the threethriving fetuses and placentas from the pregnancy identified in panel C.Notably the fetuses are smaller than normal placentas (not shown) andeven smaller than other growth-restricted placentas in panel B. Also,fetuses from this pregnancy are substantially smaller than historical,normal GD18 pups (each would normally reach ˜20 mm from nose to anus bythis gestational age).

FIG. 12. Vasopressin infusion causes dose-dependent proteinuria atGD17-GD18 in pregnant C57BL/6J female mice. (A) 24-hour urine proteinconcentration. One-way ANOVA P=0.322. (B) 24-hour urine volume. One-wayANOVA P=0.046. (C) 24-hour total urine protein. One-way ANOVA P=0.030.For all panels, * P<0.05 versus 0.24 ng/hr dose by Tukeymultiple-comparisons procedure.

FIG. 13. Vasopressin infusion causes dose-dependent intrauterine growthrestriction in pregnant C57BL/6J female mice. (A) Average fetus masses.One-way ANOVA P=0.012. (B) Average placental masses. One-way ANOVAP=0.897. For both panels, * P<0.05 versus 2.4 ng/hr dose by Tukeymultiple-comparisons procedure.

FIG. 14. Preterm (GD17) labor in 24 ng/hr vasopressin-infused C57BL/6Jmouse. On gestational day 17, one mouse that had been chronicallyinfused with vasopressin (24 ng/hr, s.c.) exhibited preterm labor. Onepup had been born and the mother had consumed the placenta and part ofthe pup before the delivery was noted by laboratory staff. The secondpup was stuck in the birth canal and required technician intervention.The animal was immediately sacrificed and photographs were obtained ofthe uterus and fetoplacental units. (A) In situ image of the uteruscontaining two developing fetuses and four partially resorbedfetoplacental units. (B) Magnified image of placentas from twodeveloping fetuses and four partially resorbed fetoplacental units. Sameruler shown in both photos; smallest division is 1 mm.

FIG. 15. Plasma vasopressin (AVP) levels in control anddouble-transgenic sRA mice. Steady-state AVP levels were unchanged incontrol littermate mice with BH4 treatment, however three days of BH4(10 mg/kg/day, i.p.) significantly (*P=0.017) reduced steady-state AVPlevels in sRA mice. Control+Vehicle n=6 (2 male, 4 female), Control+BH4n=7 (3 male, 4 female), sRA+Vehicle n=8 (3 male, 5 female), sRA+BH4 n=10(3 male, 7 female).

DESCRIPTION OF THE INVENTION

Arginine vasopressin (AVP) is a peptide hormone synthesized primarilywithin magnocellular neurons of the supraoptic nucleus andparaventricular nuclei of the brain, and this hormone is translated in a1:1 stoichiometric ratio with an inactive byproduct with a longhalf-life in the plasma, copeptin (FIG. 1). Axonal projections fromthese neurons comprise the posterior pituitary gland, and uponstimulation AVP is released into the circulation. AVP then acts uponfour major types of receptors to elicit specific effects to raise bloodpressure. V1A receptors are located within vascular smooth muscle andelicit vessel constriction. V1A receptors are also located throughoutthe central nervous system and elicit water-seeking behavior andincreased sympathetic nervous activity. V1B receptors are located withinparts of the hypothalamus and are involved in the regulation of ACTHrelease and therefore HPA-axis and glucocorticoid regulation. V2receptors are located in the collecting duct of the kidney and mobilizeaquaporin-2 to elicit water reabsorption. Finally, VACM-1 (also known asCullin-5) is involved in cell cycle regulation. Together, the primaryfunctions of AVP are thus to increase water intake, increase vascularcontraction, and increase water reabsorption with the net effect ofincreasing blood pressure.

Substantial evidence supports a causative role for AVP in thedevelopment and maintenance of hypertension in many non-pregnant models.Mice with either tightly regulated or strongly overexpressed transgenichyperactivity of the renin-angiotensin system (RAS) throughout the bodyrequire elevated AVP signaling to maintain hypertension (19, 61).Deoxycorticosterone acetate (DOCA)-salt hypertension, which is dependentupon elevated brain RAS activity (40, 50, 69) also depends upon AVPsignaling. DOCA-salt treatment results in elevated plasma AVP levels(16, 57, 60, 99). Intracerebroventricular (ICV) infusion of theangiotensin converting enzyme (ACE) inhibitor, captopril, into rats bothprevented and reversed DOCA-salt hypertension, and was associated with areduction in plasma vasopressin levels despite a reduced blood pressure(40). The dependence of DOCA-salt hypertension on AVP has also beendemonstrated using AVP-deficient Brattleboro rats, as the hypertensiveeffects of DOCA-salt are greatly diminished in these animals (16, 106).Complimenting these findings from various hypertensive models,TGR(ASrAOGEN) rats, which exhibit reduced glial production ofangiotensinogen, are hypotensive and have reduced plasma AVP levels(79). These animals also exhibit altered patterns of AVP V1A receptorexpression within the brain (11), further supporting a brain RAS-AVPinteraction. Mice deficient for the V1A AVP receptor are hypotensive,though the relative importance of brain, vascular, cardiac, thrombocyte,and hepatic receptors is unclear (2, 48). Herein, a causative role forAVP in preeclampsia has been established. Moreover, treatments forpreeclampsia targeting AVP have been identified, including inhibition ofvasopressin receptor antagonists.

Preeclampsia is a medical condition characterized by high blood pressureand significant amounts of protein in the urine of a pregnant woman. Ifleft untreated, it may develop into eclampsia, the life-threateningoccurrence of seizures during pregnancy. While blood pressure elevationmay be the most visible sign of the disease, it involves generaliseddamage to the maternal endothelium, kidneys, and liver, with the releaseof vasoconstrictive factors being a consequence of the original damage.

Preeclampsia may develop at any time after 20 weeks of gestation.Preeclampsia before 32 weeks is considered early onset, and isassociated with increased morbidity. Its progress differs amongpatients; most cases are diagnosed before labor typically would begin.Preeclampsia may also occur up to six weeks after delivery. Apart fromCaesarean section and induction of labor (and therefore delivery of theplacenta), there is no known cure. It is the most common of thedangerous pregnancy complications; it may affect both the mother andfetus.

The term “vasopressin receptor antagonist” or “VRA”, as used herein,refers to an agent which interferes with action at the vasopressinreceptors. Most commonly VRAs have been used in the treatment ofhyponatremia, especially in patients with congestive heart failure orliver cirrhosis.

VRAs may include tetracyclines or “vaptan” drugs, among others.

A tetracycline antibiotic, such as demeclocycline, may sometimes be usedto block the action of vasopressin in the kidney in hyponatremia due toinappropriately high secretion of vasopressin, when fluid restrictionhas failed.

A new class of medication, called the “vaptan” drugs, may act byinhibiting the action of vasopressin on its receptors (V1A, V1B and V2).These receptors may have a variety of functions, with the V1A and V2receptors may be expressed peripherally and involved in the modulationof blood pressure and kidney function respectively, while the V1A andV1B receptors may be expressed in the central nervous system. V1A may beexpressed in many regions of the brain, and it has been linked to avariety of social behaviors in humans and animals.

The vaptan class of drugs contains a number of compounds with varyingselectivity, several of which are either already in clinical use or inclinical trials. For example, conivaptan[N-(444,5-dihydro-2-methylimidazo[4,5-d][1]benzazepin-6(1H)-yl)carbonyl)phenyl)-(1,1′-biphenyl)-2-carboxamide;YM 087, brand name Vaprisol®] is a non-peptide VRA. It was approved in2004 for hyponatremia (low blood sodium levels) caused by syndrome ofinappropriate antidiuretic hormone, and there is some evidence it may beeffective in heart failure. Conivaptan inhibits two of the threesubtypes of the vasopressin receptor (V1a and V2). Effectively, itcauses iatrogenic nephrogenic diabetes insipidus.

Relcovaptan[1-([(2R,3S)-5-chloro-3-(2-chlorophenyl)-1-[(3,4-dimethoxyphenyl)sulfonyl]-3-hydroxy-2,3-dihydro-1H-indol-2-yl]carbonyl)-L-prolinamid;SR-49059] is a non-peptide vasopressin receptor antagonist, selectivefor the V1a subtype.

Nelivaptan[(2S,4R)-1-[(3R)-5-chloro-1-(2,4-dimethoxyphenyl)sulfonyl-3-(2-methoxyphenyl)-2-oxo-indolin-3-yl]-4-hydroxy-N,N-dimethyl-pyrrolidine-2-carboxamide]is a selective and orally active non-peptide vasopressin receptorantagonist selective for the V1b subtype.

V2 selective (V2RA) drugs may include Lixivaptan, Mozavaptan,Satavaptan, and Tolvaptan.

Lixivaptan [N-[3-chloro-4-(5H-pyrrolo-[2,1-c][1,4]benzodiazepin-;VPA-985] is a phase III pharmaceutical being developed by Cardiokine,Inc. Lixivaptan is, as of May 2010, in Phase III clinical trialsinvolving patients with hyponatremia, including those with concomitantheart failure. Hyponatremia is an electrolyte disturbance in which thesodium concentration in the serum is lower than normal. Lixivaptan mayhelp some patients eliminate excess fluids while retaining electrolytes.

Mozavaptan[N-[4-(5-Dimethylamino-2,3,4,5-tetrahydro-1-benzazepine-1-carbonyl)phenyl]-2-methylbenzamide; INN] is a vasopressin receptor antagonistmarketed by Otsuka. In Japan, it was approved in October 2006 forhyponatremia (low blood sodium levels) caused by syndrome ofinappropriate antidiuretic hormone (SIADH) due to ADH producing tumors.

Satavaptan[N-(tert-butyl)-4-[[(1s,4s)-5′-ethoxy-4-(2-morpholin-4-ylethoxy)-2′-oxospiro[cyclohexane-1,3′-indol]-1′(2′H)-yl]sulfonyl]-3-methoxybenzamide]is a vasopressin-2 receptor antagonist undergoing research for thetreatment of hyponatremia. It is also being studied for the treatment ofascites.

Tolvaptan[N-(4-[[(5R)-7-chloro-5-hydroxy-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl]carbonyl]-3-methylphenyl)-2-methylbenzamide],also known as OPC-41061, is a selective, competitive vasopressinreceptor 2 antagonist used to treat hyponatremia (low blood sodiumlevels) associated with congestive heart failure, cirrhosis, and thesyndrome of inappropriate antidiuretic hormone.

Tolvaptan is also in fast-track clinical trials[2] for polycystic kidneydisease. In a 2004 trial, tolvaptan, when administered with traditionaldiuretics, was noted to increase excretion of excess fluids and improveblood sodium levels in patients with heart failure without producingside effects such as hypotension (low blood pressure) or hypokalemia(decreased blood levels of potassium) and without having an adverseeffect on kidney function. In a recently published trial (TEMPO 3:4ClinicalTrials.gov number, NCT00428948) the study met its primary andsecondary end points. Tolvaptan, when given at an average dose of 95 mgper day over a 3-year period, slowed the usual increase in kidney volumeby 50% compared to placebo (2.80% per year versus 5.51% per year,respectively, p<0.001) and reduced the decline in kidney function whencompared with that of placebo-treated patients by approximately 30%(reciprocal serum creatinine, −2.61 versus −3.81 (mg/mL)-1 per year,p<0.001).

Tetrahydrobiopterin, “BH4,” or “THB” (trade name Kuvan) or sapropterin,refers to a naturally occurring essential cofactor of the three aromaticamino acid hydroxylase enzymes, used in the degradation of amino acidphenylalanine and in the biosynthesis of the neurotransmitters serotonin(5-hydroxytryptamine, 5-HT), melatonin, dopamine, norepinephrine(noradrenaline), epinephrine (adrenaline), and is a cofactor for theproduction of nitric oxide (NO) by the nitric oxide synthases. Thechemical name of the compound is(6R)-2-Amino-6-[(1R,2S)-1,2-dihydroxypropyl]-5,6,7,8-tetrahydropteridin-4(1H)-one.

BH4 has multiple roles in human biochemistry. One is to convert aminoacids such as phenylalanine, tyrosine, and tryptophan to precursors ofdopamine and serotonin, the body's primary neurotransmitters). BH4 alsoserves as a catalyst for the production of nitric oxide. Among otherthings, nitric oxide is involved in vasodilation, which improvessystematic blood flow. The role of BH4 in this enzymatic process is socritical that some research points to a deficiency of BH4—and thus, ofnitric oxide—as being a core cause of the neurovascular dysfunction thatis the hallmark of circulation-related diseases such as diabetes.

The term “chemically related compound,” as used herein in the context ofBH4, refers to a compound having similar chemical structure to that ofBH4. For example, a chemically related compound may have any othersuitable substitution group instead of the amino group in the aromaticring of BH4. A chemically related compound may also have any othersuitable substitution groups on any other location of the BH4.

The term “extracorporeal therapy,” as used herein, refers to anextracorporeal medical procedure or a medical procedure which isperformed outside the body. An exemplary extracorporeal therapy mayinclude circulatory procedures. Circulatory procedures are procedures inwhich blood is taken from a patient's circulation to have a processapplied to it before it is returned to the circulation. All of theapparatus carrying the blood outside the body is termed theextracorporeal circuit.

The present disclosure contemplates pharmaceutical formulations, dosageforms, kits, and methods wherein a compound, such as a pharmaceuticalcompound, that reverses or negates the effects of AVP or mimics thereofto reduce preeclampsia in a subject in need thereof.

The contemplated pharmaceutical formulations, dosage forms, kits, andmethods may further include a plurality of drugs or pharmaceuticallyacceptable salts or derivatives thereof that inhibit production and/orsecretion of AVP and/or AVP receptors together with one or morepharmaceutically acceptable carriers therefor, and optionally, othertherapeutic and/or prophylactic ingredients. The carrier(s) should beacceptable in the sense of being compatible with the other ingredientsof the formulation and being physiologically acceptable to the recipientthereof.

For example, compositions herein may be formulated for oral, rectal,nasal, topical (including buccal and sublingual), transdermal, vaginal,injection/injectable, and/or parental (including subcutaneous,intramuscular, intravenous, and intradermal) administration. Othersuitable administration routes are incorporated herein. The compositionsmay be presented conveniently in unit dosage forms and may be preparedby any methods known in the pharmaceutical arts. Examples of suitabledrug formulations and/or forms are discussed in, for example, Hoover,John E. Remington's Pharmaceutical Sciences, Mack Publishing Co., Eston,Pa.; 18.sup.th edition (1995); and Liberman, H. A. and Lachman, L. Eds.,Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980.Illustrative methods include the step of bringing one or more activeingredients into association with a carrier that constitutes one or moreaccessory ingredients. In general, the compositions may be prepared bybringing into association uniformly and intimately one or more activeingredients with liquid carriers or finely divided solid carriers orboth, and then, if necessary, shaping the product.

Pharmaceutical formulations may include those suitable for oral,intramuscular, rectal, nasal, topical (including buccal andsub-lingual), vaginal or parenteral (including intramuscular,subcutaneous and intravenous) administration or in a form suitable foradministration by inhalation or insufflation. One or more of thecompounds of the invention, together with a conventional adjuvant,carrier, or diluent, may thus be placed into the form of pharmaceuticalcompositions and unit dosages thereof, and in such form may be employedas solids, such as tablets or filled capsules, or liquids such assolutions, suspensions, emulsions, elixirs, or capsules filled with thesame, all for oral use, in the form of suppositories for rectaladministration; or in the form of sterile injectable solutions forparenteral (including subcutaneous) use. Such pharmaceuticalcompositions and unit dosage forms thereof may comprise conventionalingredients in conventional proportions, with or without additionalactive compounds or principles, and such unit dosage forms may containany suitable effective amount of the active ingredient commensurate withthe intended daily dosage range to be employed.

A salt may be a pharmaceutically suitable (i.e., pharmaceuticallyacceptable) salt including, but not limited to, acid addition saltsformed by mixing a solution of the instant compound with a solution of apharmaceutically acceptable acid. A pharmaceutically acceptable acid maybe, for example, hydrochloric acid, methanesulphonic acid, fumaric acid,maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid,citric acid, tartaric acid, carbonic acid or phosphoric acid.

Suitable pharmaceutically-acceptable salts may further include, but arenot limited to salts of pharmaceutically-acceptable inorganic acids,including, for example, sulfuric, phosphoric, nitric, carbonic, boric,sulfamic, and hydrobromic acids, or salts of pharmaceutically-acceptableorganic acids such propionic, butyric, maleic, hydroxymaleic, lactic,mucic, gluconic, benzoic, succinic, phenylacetic, toluenesulfonic,benezenesulfonic, salicyclic sulfanilic, aspartic, glutamic, edetic,stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic, andvaleric acids.

Various pharmaceutically acceptable salts include, for example, the listof FDA-approved commercially marketed salts including acetate,benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calciumedetate, camsylate, carbonate, chloride, citrate, dihydrochloride,edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate,glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine,hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate,lactate, lactobionate, malate, maleate, mandelate, mesylate,methylbromide, methylnitrate, methylsulfate, mucate, napsylate, mitrate,pamoate, pantothenate, phosphate, diphosphate, polygalacturonate,salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate,teoclate, and triethiodide.

A hydrate may be a pharmaceutically suitable (i.e., pharmaceuticallyacceptable) hydrate that is a compound formed by the addition of wateror its elements to a host molecule (for example, the free form versionof the compound) including, but not limited to, monohydrates,dihydrates, etc.

A solvate may be a pharmaceutically suitable (i.e., pharmaceuticallyacceptable) solvate, whereby solvation is an interaction of a solutewith a solvent which leads to stabilization of the solute species in asolution, and whereby the solvated state is an ion in a solutioncomplexed by solvent molecules. Solvates and hydrates may also bereferred to as “analogues.”

A prodrug may be a compound that is pharmacologically inert but areconverted by enzyme or chemical action to an active form of the drug(i.e., an active pharmaceutical ingredient) at or near the predeterminedtarget site. In other words, prodrugs are inactive compounds that yieldan active compound upon metabolism in the body, which may or may not beenzymatically controlled. Prodrugs may also be broadly classified intotwo groups: bioprecursor and carrier prodrugs. Prodrugs may also besubclassified according to the nature of their action. Bioprecursorprodrugs are compounds that already contain the embryo of the activespecies within their structure, whereby the active species are producedupon metabolism.

Carrier prodrugs are formed by combining the active drug with a carrierspecies forming a compound having desirable chemical and biologicalcharacteristics, whereby the link is an ester or amide so that thecarrier prodrug is easily metabolized upon absorption or delivery to thetarget site. For example, lipophilic moieties may be incorporated toimprove transport through membranes. Carrier prodrugs linked by afunctional group to carrier are referred to as bipartite prodrugs.Prodrugs where the carrier is linked to the drug by a separate structureare referred to as tripartite prodrugs, whereby the carrier is removedby an enzyme-controlled metabolic process, and whereby the linkingstructure is removed by an enzyme system or by a chemical reaction. Ahydroxy-protecting group includes, for example, a tert-butyloxy-carbonyl(t-BOC) and t-butyl-dimethyl-silyl (TBS). Other hydroxy protectinggroups contemplated are known in the art.

In another embodiment, a dosage form and/or composition may include oneor more active metabolites of the active ingredients in place of or inaddition to the active ingredients disclosed herein. Dosage formcompositions containing the active pharmaceutical ingredients may alsocontain one or more inactive pharmaceutical ingredients such asdiluents, solubilizers, alcohols, binders, controlled release polymers,enteric polymers, disintegrants, excipients, colorants, flavorants,sweeteners, antioxidants, preservatives, pigments, additives, fillers,suspension agents, surfactants (for example, anionic, cationic,amphoteric and nonionic), and the like. Various FDA-approved topicalinactive ingredients are found at the FDA's “The Inactive IngredientsDatabase” that contains inactive ingredients specifically intended assuch by the manufacturer, whereby inactive ingredients can also beconsidered active ingredients under certain circumstances, according tothe definition of an active ingredient given in 21 CFR 210.3(b)(7).Alcohol is a good example of an ingredient that may be considered eitheractive or inactive depending on the product formulation.

As used herein, a kit may be a packaged collection of related materials,including, for example, a single and/or a plurality of dosage forms eachapproximating an effective amount of an active ingredient, such as, forexample, an AVP receptor inhibitor and/or an additional drug. Theincluded dosage forms may be taken at one time, or at prescribedinterval.

As used herein, an oral dosage form may include capsules (a solid oraldosage form consisting of a shell and a filling, whereby the shell iscomposed of a single sealed enclosure, or two halves that fit togetherand which are sometimes sealed with a band and whereby capsule shellsmay be made from gelatin, starch, or cellulose, or other suitablematerials, may be soft or hard, and are filled with solid or liquidingredients that can be poured or squeezed), capsule or coated pellets(solid dosage form in which the drug is enclosed within either a hard orsoft soluble container or “shell” made from a suitable form of gelatin;the drug itself is in the form of granules to which varying amounts ofcoating have been applied), capsule coated extended release (a soliddosage form in which the drug is enclosed within either a hard or softsoluble container or “shell” made from a suitable form of gelatin;additionally, the capsule is covered in a designated coating, and whichreleases a drug or drugs in such a manner to allow at least a reductionin dosing frequency as compared to that drug or drugs presented as aconventional dosage form), capsule delayed release (a solid dosage formin which the drug is enclosed within either a hard or soft solublecontainer made from a suitable form of gelatin, and which releases adrug (or drugs) at a time other than promptly after administration,whereby enteric-coated articles are delayed release dosage forms),capsule delayed release pellets (solid dosage form in which the drug isenclosed within either a hard or soft soluble container or “shell” madefrom a suitable form of gelatin); the drug itself is in the form ofgranules to which enteric coating has been applied, thus delayingrelease of the drug until its passage into the intestines), capsuleextended release (a solid dosage form in which the drug is enclosedwithin either a hard or soft soluble container made from a suitable formof gelatin, and which releases a drug or drugs in such a manner to allowa reduction in dosing frequency as compared to that drug or drugspresented as a conventional dosage form), capsule film-coated extendedrelease (a solid dosage form in which the drug is enclosed within eithera hard or soft soluble container or “shell” made from a suitable form ofgelatin; additionally, the capsule is covered in a designated filmcoating, and which releases a drug or drugs in such a manner to allow atleast a reduction in dosing frequency as compared to that drug or drugspresented as a conventional dosage form), capsule gelatin coated (asolid dosage form in which the drug is enclosed within either a hard orsoft soluble container made from a suitable form of gelatin; through abanding process, the capsule is coated with additional layers of gelatinso as to form a complete seal), and capsule liquid filled (a soliddosage form in which the drug is enclosed within a soluble, gelatinshell which is plasticized by the addition of a polyol, such as sorbitolor glycerin, and is therefore of a somewhat thicker consistency thanthat of a hard shell capsule; typically, the active ingredients aredissolved or suspended in a liquid vehicle).

Oral dosage forms contemplated herein also include granules (a smallparticle or grain), pellet (a small sterile solid mass consisting of ahighly purified drug, with or without excipients, made by the formationof granules, or by compression and molding), pellets coated extendedrelease (a solid dosage form in which the drug itself is in the form ofgranules to which varying amounts of coating have been applied, andwhich releases a drug or drugs in such a manner to allow a reduction indosing frequency as compared to that drug or drugs presented as aconventional dosage form), pill (a small, round solid dosage formcontaining a medicinal agent intended for oral administration), powder(an intimate mixture of dry, finely divided drugs and/or chemicals thatmay be intended for internal or external use), elixir (a clear,pleasantly flavored, sweetened hydroalcoholic liquid containingdissolved medicinal agents; it is intended for oral use), chewing gum (asweetened and flavored insoluble plastic material of various shapeswhich when chewed, releases a drug substance into the oral cavity), orsyrup (an oral solution containing high concentrations of sucrose orother sugars; the term has also been used to include any other liquiddosage form prepared in a sweet and viscid vehicle, including oralsuspensions).

Oral dosage forms contemplated herein may further include a tablet (asolid dosage form containing medicinal substances with or withoutsuitable diluents), tablet chewable (a solid dosage form containingmedicinal substances with or without suitable diluents that is intendedto be chewed, producing a pleasant tasting residue in the oral cavitythat is easily swallowed and does not leave a bitter or unpleasantafter-taste), tablet coated (a solid dosage form that contains medicinalsubstances with or without suitable diluents and is covered with adesignated coating), tablet coated particles (a solid dosage formcontaining a conglomerate of medicinal particles that have each beencovered with a coating), tablet delayed release (a solid dosage formwhich releases a drug or drugs at a time other than promptly afteradministration, whereby enteric-coated articles are delayed releasedosage forms), tablet delayed release particles (a solid dosage formcontaining a conglomerate of medicinal particles that have been coveredwith a coating which releases a drug or drugs at a time other thanpromptly after administration, whereby enteric-coated articles aredelayed release dosage forms), tablet dispersible (a tablet that, priorto administration, is intended to be placed in liquid, where itscontents will be distributed evenly throughout that liquid, whereby term‘tablet, dispersible’ is no longer used for approved drug products, andit has been replaced by the term ‘tablet, for suspension’), tableteffervescent (a solid dosage form containing mixtures of acids, forexample, citric acid, tartaric acid, and sodium bicarbonate, whichrelease carbon dioxide when dissolved in water, whereby it is intendedto be dissolved or dispersed in water before administration), tabletextended release (a solid dosage form containing a drug which allows atleast a reduction in dosing frequency as compared to that drug presentedin conventional dosage form), tablet film coated (a solid dosage formthat contains medicinal substances with or without suitable diluents andis coated with a thin layer of a water-insoluble or water-solublepolymer), tablet film coated extended release (a solid dosage form thatcontains medicinal substances with or without suitable diluents and iscoated with a thin layer of a water-insoluble or water-soluble polymer;the tablet is formulated in such manner as to make the containedmedicament available over an extended period of time followingingestion), tablet for solution (a tablet that forms a solution whenplaced in a liquid), tablet for suspension (a tablet that forms asuspension when placed in a liquid, which is formerly referred to as a‘dispersible tablet’), tablet multilayer (a solid dosage form containingmedicinal substances that have been compressed to form amultiple-layered tablet or a tablet-within-a-tablet, the inner tabletbeing the core and the outer portion being the shell), tablet multilayerextended release (a solid dosage form containing medicinal substancesthat have been compressed to form a multiple-layered tablet or atablet-within-a-tablet, the inner tablet being the core and the outerportion being the shell, which, additionally, is covered in a designatedcoating; the tablet is formulated in such manner as to allow at least areduction in dosing frequency as compared to that drug presented as aconventional dosage form), tablet orally disintegrating (a solid dosageform containing medicinal substances which disintegrates rapidly,usually within a matter of seconds, when placed upon the tongue), tabletorally disintegrating delayed release (a solid dosage form containingmedicinal substances which disintegrates rapidly, usually within amatter of seconds, when placed upon the tongue, but which releases adrug or drugs at a time other than promptly after administration),tablet soluble (a solid dosage form that contains medicinal substanceswith or without suitable diluents and possesses the ability to dissolvein fluids), tablet sugar coated (a solid dosage form that containsmedicinal substances with or without suitable diluents and is coatedwith a colored or an uncolored water-soluble sugar), and the like.

Injection and infusion dosage forms (i.e., parenteral dosage forms)include, but are not limited to, the following. Liposomal injectionincludes or forms liposomes or a lipid bilayer vesicle havingphospholipids that encapsulate an active drug substance. Injectionincludes a sterile preparation intended for parenteral use. Fivedistinct classes of injections exist as defined by the USP. Emulsioninjection includes an emulsion comprising a sterile, pyrogen-freepreparation intended to be administered parenterally. Lipid complex andpowder for solution injection are sterile preparations intended forreconstitution to form a solution for parenteral use.

Powder for suspension injection is a sterile preparation intended forreconstitution to form a suspension for parenteral use. Powderlyophilized for liposomal suspension injection is a sterile freeze driedpreparation intended for reconstitution for parenteral use that isformulated in a manner allowing incorporation of liposomes, such as alipid bilayer vesicle having phospholipids used to encapsulate an activedrug substance within a lipid bilayer or in an aqueous space, wherebythe formulation may be formed upon reconstitution. Powder lyophilizedfor solution injection is a dosage form intended for the solutionprepared by lyophilization (“freeze drying”), whereby the processinvolves removing water from products in a frozen state at extremely lowpressures, and whereby subsequent addition of liquid creates a solutionthat conforms in all respects to the requirements for injections. Powderlyophilized for suspension injection is a liquid preparation intendedfor parenteral use that contains solids suspended in a suitable fluidmedium, and it conforms in all respects to the requirements for SterileSuspensions, whereby the medicinal agents intended for the suspensionare prepared by lyophilization.

Solution injection involves a liquid preparation containing one or moredrug substances dissolved in a suitable solvent or mixture of mutuallymiscible solvents that is suitable for injection. Solution concentrateinjection involves a sterile preparation for parenteral use that, uponaddition of suitable solvents, yields a solution suitable forinjections. Suspension injection involves a liquid preparation (suitablefor injection) containing solid particles dispersed throughout a liquidphase, whereby the particles are insoluble, and whereby an oil phase isdispersed throughout an aqueous phase or vice-versa. Suspensionliposomal injection is a liquid preparation (suitable for injection)having an oil phase dispersed throughout an aqueous phase in such amanner that liposomes (a lipid bilayer vesicle usually containingphospholipids used to encapsulate an active drug substance either withina lipid bilayer or in an aqueous space) are formed. Suspension sonicatedinjection is a liquid preparation (suitable for injection) containingsolid particles dispersed throughout a liquid phase, whereby theparticles are insoluble. In addition, the product may be sonicated as agas is bubbled through the suspension resulting in the formation ofmicrospheres by the solid particles.

A parenteral carrier system may include one or more pharmaceuticallysuitable excipients, such as solvents and co-solvents, solubilizingagents, wetting agents, suspending agents, thickening agents,emulsifying agents, chelating agents, buffers, pH adjusters,antioxidants, reducing agents, antimicrobial preservatives, bulkingagents, protectants, tonicity adjusters, and special additives.

Inhalation dosage forms include, but are not limited to, aerosol being aproduct that is packaged under pressure and contains therapeuticallyactive ingredients that are released upon activation of an appropriatevalve system intended for topical application to the skin as well aslocal application into the nose (nasal aerosols), mouth (lingual andsublingual aerosols), or lungs (inhalation aerosols). Inhalation dosageforms further include foam aerosol being a dosage form containing one ormore active ingredients, surfactants, aqueous or nonaqueous liquids, andthe propellants, whereby if the propellant is in the internal(discontinuous) phase (i.e., of the oil-in-water type), a stable foam isdischarged, and if the propellant is in the external (continuous) phase(i.e., of the water-in-oil type), a spray or a quick-breaking foam isdischarged. Inhalation dosage forms also include metered aerosol being apressurized dosage form consisting of metered dose valves which allowfor the delivery of a uniform quantity of spray upon each activation;powder aerosol being a product that is packaged under pressure andcontains therapeutically active ingredients, in the form of a powder,that are released upon activation of an appropriate valve system; andaerosol spray being an aerosol product which utilizes a compressed gasas the propellant to provide the force necessary to expel the product asa wet spray and being applicable to solutions of medicinal agents inaqueous solvents.

Pharmaceutically suitable inhalation carrier systems may includepharmaceutically suitable inactive ingredients known in the art for usein various inhalation dosage forms, such as (but not limited to) aerosolpropellants (for example, hydrofluoroalkane propellants), surfactants,additives, suspension agents, solvents, stabilizers and the like.

A transdermal dosage form may include, but is not limited to, a patchbeing a drug delivery system that often contains an adhesive backingthat is usually applied to an external site on the body, whereby theingredients either passively diffuse from, or are actively transportedfrom some portion of the patch, and whereby depending upon the patch,the ingredients are either delivered to the outer surface of the body orinto the body; and other various types of transdermal patches such asmatrix, reservoir and others known in the art. The “pharmaceuticallysuitable transdermal carrier system” includes pharmaceutically suitableinactive ingredients known in the art for use in various transdermaldosage forms, such as (but not limited to) solvents, adhesives,diluents, additives, permeation enhancing agents, surfactants,emulsifiers, liposomes, and the like.

Suitable dosage amounts and dosing regimens may be selected inaccordance with a variety of factors, including one or more particularconditions being treated, the severity of the one or more conditions,the genetic profile, age, health, sex, diet, and weight of the subject,the route of administration alone or in combination with pharmacologicalconsiderations including the activity, efficacy, bioavailability,pharmacokinetic, and toxicological profiles of the particular compoundemployed, whether a drug delivery system is utilized and whether thedrug is administered as part of a drug combination. Therefore, thedosage regimen to be employed may vary widely and may necessarilydeviate from the dosage regimens set forth herein.

Contemplated dosage forms may include an amount of one or more compoundsthat inhibit production and/or secretion of AVP and/or inhibit orotherwise block AVP receptors or the effects of AVP ranging from about 1to about 1200 mg/kg, or about 1 to about 50 mg/kg, or about 5 to about100 mg/kg, or about 25 to about 800 mg/kg, or about 100 to about 500mg/kg, or 0.1 to 50 milligrams/kilogram (±10%), or 10 to 100milligrams/kilogram (±10%), or 1 to 600 milligrams/kilogram (±10%), or0.1 to 200 milligrams/kilogram (±10%), or 1 to 100 milligrams/kilogram(±10%), or 5 to 50 milligrams/kilogram (±10%), or 30 milligrams/kilogram(±10%), or 20 milligrams/kilogram (±10%), or 10 milligrams/kilogram(±10%), or 5 milligrams/kilogram (±10%), per dosage form, such as, forexample, a tablet, a pill, a bolus, and the like.

In another embodiment, a dosage form may be administered to a subject inneed thereof once per day, or twice per day, or once every 6 hours, oronce every 4 hours, or once every 2 hours, or hourly, or twice an hour,or twice a day, or twice a week, or monthly. A therapeutically effectiveamount of a compound that inhibits production and/or secretion and/oreffects of AVP and/or AVP receptors or mimics thereof, such as, forexample, a “vaptan,” may be any amount that begins to reducepreeclampsia features in a subject receiving the compound.

It is further contemplated that one active ingredient may be in anextended release form, while a second other may not be, so the recipientexperiences, for example, a spike in the second active ingredient thatdissipates rapidly, while the first active ingredient is maintained in ahigher concentration in the blood stream over a longer period of time.Similarly, one of the active ingredients may be an active metabolite,while another may be in an unmetabolized state, such that the activemetabolite has an immediate effect upon administration to a subjectwhereas the unmetabolized active ingredient administered in a singledosage form may need to be metabolized before taking effect in thesubject.

Also contemplated are solid form preparations that include at least oneactive ingredient which are intended to be converted, shortly beforeuse, to liquid form preparations for oral administration. Such liquidforms include solutions, suspensions, and emulsions. These preparationsmay contain, in addition to the active component, colorants, flavors,stabilizers, buffers, artificial and natural sweeteners, dispersants,thickeners, solubilizing agents, and the like.

Solutions or suspensions may be applied topically and/or directly to thenasal cavity, respiratory tract, eye, or ear by conventional means, forexample with a dropper, pipette or spray. Alternatively, one or more ofthe active ingredients may be provided in the form of a dry powder, forexample a powder mix of the compound in a suitable powder base such aslactose, starch, starch derivatives such as hydroxypropylmethylcellulose and polyvinylpyrrolidone (PVP). Conveniently the powdercarrier may form a gel in the nasal cavity. The powder composition maybe presented in unit dose form, for example, in capsules or cartridgesof, for example, gelatin, or blister packs from which the powder may beadministered by means of an inhaler.

The pharmaceutical preparations may be in unit dosage forms. In suchform, the preparation may be subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, such as a kit or other form, the packagecontaining discrete quantities of preparation, such as packeted tablets,capsules, liquids or powders in vials or ampules. Also, the unit dosageform can be a capsule, tablet, cachet, or lozenge, or it can be theappropriate number of any of these in packaged form. Contemplated kitsmay include any combination of disclosed dosage forms.

In one embodiment, a method for the treatment of preeclampsia in asubject in need thereof includes administering to the subject atherapeutically effective amount of a pharmaceutical compound thatinhibits an arginine vasopressin receptor.

In one embodiment, the amount of the pharmaceutical compound is at leastabout 1 to about 50 mg per day.

In another embodiment, the arginine vasopressin receptor includes atleast one of V1A, V2, and V1B.

In one embodiment, the pharmaceutical compound is a vasopressin receptorantagonist. Examples of vasopressin receptor antagonists include“vaptan” drugs. Specific examples of vaptans includes conivaptan,tolvaptan, and relcovaptan, lixivaptan, mozavaptan, satavaptan. It isenvisioned that combinations of vaptans and/or or additional vasopressinreceptor antagonists may be combined into a single composition. It isfurther envisioned that drugs that inhibit secretion and/or productionof AVP may be combined with vasopressin receptor antagonists to form atherapeutic composition for the treatment of preeclampsia.

In one example, a composition for treatment of preeclampsia includes atherapeutically effective amount of a first arginine vasopressinreceptor inhibitor and optionally a therapeutically effective amount ofa second arginine vasopressin receptor inhibitor. For example, acomposition may include a first arginine vasopressin receptor inhibitor,such as conivaptan, and/or a second arginine vasopressin receptorinhibitor and/or an inhibitor of AVP secretion and/or production.

In another embodiment, a composition for treatment of preeclampsia maycomprise a therapeutically effective amount of tetrahydrobiopterin (BH4)or a chemically related compound.

In one embodiment, a composition for treatment of preeclampsia maycomprise a therapeutically effective amount of BH4 or a chemicallyrelated compound, and a therapeutically effective amount of at least onearginine vasopressin receptor inhibitor as discussed above or anyinhibitors as understood by a person having ordinary skill in the art.

In one embodiment, a composition for treatment of preeclampsia maycomprise a therapeutically effective amount of BH4 or a chemicallyrelated compound, a therapeutically effective amount of a first argininevasopressin receptor inhibitor and optionally a therapeuticallyeffective amount of a second arginine vasopressin receptor inhibitor.For example, a composition may comprise BH4 or a chemically relatedcompound, a first arginine vasopressin receptor inhibitor, such asconivaptan, and/or a second arginine vasopressin receptor inhibitorand/or an inhibitor of AVP secretion and/or production.

In a third aspect, a pharmaceutical dosage form includes atherapeutically effective amount of a first arginine vasopressinreceptor inhibitor, a therapeutically effective amount of a secondarginine vasopressin receptor inhibitor, and one or morepharmaceutically suitable carriers, diluent, and/or excipients.

In one embodiment, the dosage form includes an oral, injection,infusion, inhalation, transdermal, or implant dosage form.

In another embodiment, the pharmaceutical dosage form includes about 1to about 500 mg of conivaptan and about 1 to about 500 mg of at leastone of tolvaptan and relcovaptan.

The treatments contemplated herein may be administered prophylacticallybefore onset of preeclampsia, as well as after onset of preeclampsia. Itis further envisioned that treatment regimens (e.g., dosing levels) mayalter over time and may be informed by urine and/or serum measurementsof vasopressin, neurophysin II, and/or copeptin levels.

In another aspect, a pharmaceutical dosage form may comprise atherapeutically effective amount of BH4 or a chemically related compoundand one or more pharmaceutically suitable carriers, diluents, and/orexcipients.

In one embodiment, a pharmaceutical dosage form may comprise atherapeutically effective amount of BH4 or a chemically related compoundand a therapeutically effective amount of at least one argininevasopressin receptor inhibitor. A suitable arginine vasopressin receptorinhibitor may include those as discussed above and any inhibitors asunderstood by a person having ordinary skill in the art.

In one specific embodiment, the arginine vasopressin receptor inhibitormay comprise at least one of conivaptan, tolvaptan and relcovaptan.

In one embodiment, a pharmaceutical dosage form may comprise atherapeutically effective amount of BH4 or a chemically relatedcompound, a first arginine vasopressin receptor inhibitor, atherapeutically effective amount of a second arginine vasopressinreceptor inhibitor, and one or more pharmaceutically suitable carriers,diluent, and/or excipients.

In one specific embodiment, the arginine vasopressin receptor inhibitormay comprise at least one of conivaptan, tolvaptan and relcovaptan.

An appropriate dosage level for the present invention may generally beabout 0.001 to about 500 mg per kg subject body weight per day which canbe administered in a single or multiple doses. Preferably, the dosagelevel will be about 0.01 to about 250 mg/kg per day; more preferablyabout 0.05 to about 100 mg/kg per day. A suitable dosage level may beabout 0.01 to about 250 mg/kg per day, about 0.05 to about 100 mg/kg perday, or about 0.1 to about 50 mg/kg per day. Within this range thedosage may be about 0.05 to about 0.5, about 0.5 to about 5 or about 5to about 50 mg/kg per day. The dosage may be selected, for example, toinclude any dose within any of these ranges, for therapeutic efficacyand/or symptomatic adjustment of the dosage to the subject to betreated.

Dosage ranges for agents may be as low as 5 ng/day. In certainembodiments, about 10 ng/day, about 15 ng/day, about 20 ng/day, about 25ng/day, about 30 ng/day, about 35 ng/day, about 40 ng/day, about 45ng/day, about 50 ng/day, about 60 ng/day, about 70 ng/d, about 80ng/day, about 90 ng/day, about 100 ng/day, about 200 ng/day, about 300ng/day, about 400 ng/day, about 500 ng/day, about 600 ng/day, about 700ng/day, about 800 ng/day, about 900 ng/day, about 1 μg/day, about 2μg/day, about 3 μg/day, about 4 μg/day, about 5 μg/day, about 10 μg/day,about 15 μg/day, about 20 μg/day, about 30 μg/day, about 40 μg/day,about 50 μg/day, about 60 μg/day, about 70 μg/day, about 80 μg/day,about 90 μg/day, about 100 μg/day, about 200 μg/day, about 300 μg/day,about 400 μg/day, about 500 μg/day, about 600 μg/day, about 700 μg/day,about 800 μg/day, about 900 μg/day, about 1 mg/day, about 2 mg/day,about 3 mg/day, about 4 mg/day, about 5 mg/day, about 10 mg/day, about15 mg/day, about 20 mg/day, about 30 mg/day, about 40 mg/day, or about50 mg/day of an agent of the invention is administered.

In certain embodiments, the agents of the invention are administered inpM or nM concentrations. In certain embodiments, the agents areadministered in about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, about 10 pM, about20 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM,about 80 pM, about 90 pM, about 100 pM, about 200 pM, about 300 pM,about 400 pM, about 500 pM, about 600 pM, about 700 pM, about 800 pM,about 900 pM, about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM,about 80 nM, about 90 nM, about 100 nM, about 200 nM, about 300 nM,about 400 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, orabout 900 nM concentrations.

In certain embodiments, the size of the active agent is important. Incertain embodiments, the active agent is less than about 3 μm, less thanabout 2 μm, less than about 1 μm in diameter. In certain embodiments,the active agent is from about 0.1 μm to about 3.0 μm in diameter. Incertain embodiments, the active agent is from about 0.5 μm to about 1.5μm in diameter. In certain embodiments, the active agent is about 0.2μm, about 0.3 μm, about 0.4 μm, about 0.5 μm, about 0.6 μm, about 0.7μm, about 0.8 μm, about 0.9 μm, about 1.0 μm, about 1.1 μm, about 1.2μm, about 1.3 μm, about 1.4 μm, or about 1.5 μm in diameter.

It may be advantageous for the pharmaceutical combination to becomprised of a relatively large amount of the first component comparedto the second component. In certain instances, the ratio of the firstactive agent to second active agent is about 200:1, 190:1, 180:1, 170:1,160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, 90:1, 80:1, 70:1, 60:1,50:1, 40:1, 30:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1,2:1, or 1:1. It further may be preferable to have a more equaldistribution of pharmaceutical agents. In certain instances, the ratioof the first active agent to the second active agent is about 4:1, 3:1,2:1, 1:1, 1:2, 1:3, or 1:4. It also may be advantageous for thepharmaceutical combination to have a relatively large amount of thesecond component compared to the first component. In certain instances,the ratio of the second active agent to the first active agent is about30:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, or 5:1. In certaininstances, the ratio of the second active agent to first active agent isabout 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, or 40:1. In certaininstances, the ratio of the second active agent to first active agent isabout 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, or110:1. A composition comprising any of the above-identified combinationsmay be administered in divided doses about 1, 2, 3, 4, 5, 6, or moretimes per day or in a form that will provide a rate of release effectiveto attain the desired results. The dosage form may contain both thefirst and second active agents. The dosage form may be administered onetime per day if it contains both the first and second active agents.

For example, a formulation intended for oral administration to humansmay contain from about 0.1 mg to about 5 g of the first therapeuticagent and about 0.1 mg to about 5 g of the second therapeutic agent,both of which are compounded with an appropriate and convenient amountof carrier material varying from about 5 to about 95 percent of thetotal composition. Unit dosages will generally contain between about 0.5mg to about 1500 mg of the first therapeutic agent and 0.5 mg to about1500 mg of the second therapeutic agent. The dosage may be about 25 mg,50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, or 1000mg, etc., up to about 1500 mg of the first therapeutic agent. The dosagemay be about 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600mg, 800 mg, or 1 000 mg, etc., up to about 1500 mg of the secondtherapeutic agent.

In one aspect, a method of treating preeclampsia in a subject in needthereof is disclosed. The present method of treating preeclampsia in asubject may comprise the step of removing vasopressin from thecirculation system (e.g., blood) of a patient. In one embodiment, themethod may comprise the steps of a) removing whole blood from a patient;b) separating the blood into individual components; c) reducing orremoving vasopressin in the blood; and d) re-circulating the remainingblood components into the bloodstream of the patient. In one embodiment,the present method of treating preeclampsia may be applied by using anysuitable apparatus. For example, one may use any commercially availableextracorporeal therapy techniques, such as apheresis, hemodialysis (alsohaemodialysis), hemofiltration, and others. For example, column-basedmodulation of vasopressin may be used as part of a multifaceted orsingular treatment or prevention for preeclampsia focused on decreasingcirculating vasopressin. An extracorporeal apheresis type column may beused to this end. Similar to apheresis columns used in renalhemodialysis, in this embodiment, a column may be used to pass humanblood through that: 1) chelates and/or absorbs circulating vasopressinand/or copeptin using a chelator of vasopressin, such as but not limitedto, an antibody that is attached to the stationary phase of the columnand/or internal column surfaces; 2) inactivates circulating vasopressinand/or copeptin using a column loaded with LNPEP and/or vasopressinase;3) simultaneously inactivates or chelates other effectors ofpreeclampsia such as sFLT-1; and 4) all of the above.

Much like hemodialysis, treatment using these developed columns wouldinvolve placing an intravenous (IV) line into a patient which would pumpthe patient blood extracorporeally through the IV line into thedeveloped column. The column chelates or otherwise inactivatesvasopressin and/or copeptin. The blood will then be pumped back into thepatient to improve the subject's health.

There are currently preliminary data to suggest that extracorporealremoval of sFLT-1 is possible using column technology. In a pilot studyby Thadhani, et al. (93), five women with very preterm preeclampsiaunderwent a single dextran sulfate cellulose apheresis treatment. Inthese women, the treatment decreased circulating sFlt-1, reducedproteinuria, and stabilized blood pressure without adverse effects tothe mother and fetus. A developed extracorporeal column to chelateand/or inactivate vasopressin and/or copeptin can be used in a similarway.

Unless defined otherwise in this specification, technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs and byreference to published texts.

It should be understood that while various embodiments in thespecification are presented using “comprising” language, under variouscircumstances, a related embodiment may also be described using“consisting of” or “consisting essentially of” language. It is to benoted that the term “a” or “an,” refers to one or more, for example, “animmunoglobulin molecule,” is understood to represent one or moreimmunoglobulin molecules. As such, the terms “a” (or “an”), “one ormore,” and “at least one” is used interchangeably herein.

The following examples set forth preferred markers and methods inaccordance with the invention. It is to be understood, however, thatthese examples are provided by way of illustration and nothing hereinshould be taken as a limitation upon the overall scope of the invention.

EXAMPLES

The following examples illustrate that vasopressin infusionrecapitulates all in one model, simultaneously, all of the keyphenotypes of preeclampsia: increased blood pressure, renalmorphological pathologies, proteinuria, intrauterine growth restriction,and spontaneous preterm labor. Therefore, these examples establish acausative role of vasopressin in preeclampsia and have enabled theidentification of tools and agents for the successful treatment ofpreeclampsia.

Example 1. Hypertension in Mice with Transgenic Activation of the BrainRenin-Angiotensin System is Vasopressin Dependent

Activity of the local tissue renin-angiotensin system (RAS) within thebrain has been implicated in the development and maintenance of elevatedblood pressure in many forms of hypertension. Evidence specificallydemonstrating a causal role for brain RAS activity in hypertension comesfrom various rodent models. These many models include peripheralangiotensin infusion models (63, 82, 107), both elevated (20) andsuppressed (38, 50, 69) plasma renin models, psychogenic (59), coldexposure (88), renal injury (103), sleep apnea (18) models, transgenicTGR (mRen2)27 rats (89), and both Dahl salt-sensitive (38) andspontaneously hypertensive rats (SHR) maintained on high-salt diets (49,65, 102). Two major mechanisms have been documented that account for theblood pressure effects of brain angiotensin. First, actions of the RASwithin the supraoptic (SON) and paraventricular hypothalamic nuclei(PVN) stimulate the production and release of arginine vasopressin (AVP,also known as antidiuretic hormone, ADH, or argipressin) (6, 15, 23, 40,70, 73, 89). Second, hindbrain and brain stem actions of the RAS alterbaroreflex function and sympathetic output (30, 34). Interestingly, apopulation of AVP-expressing neurons project from the PVN to thehindbrain and spinal cord and appear to be involved in the regulation ofsympathetic nervous activity, suggesting a possible AVP-mediatedcross-talk between these two mechanisms.

Although some studies have failed to document a substantial role for AVPin blood pressure control in heterogenous groups of human subjects (67),AVP has been implicated as a significant contributor to blood pressurecontrol in selected populations of humans (26, 66). Specifically,African Americans (4), the elderly (21), and patients with congestiveheart failure (26) or chronic renal failure (3) all exhibitAVP-dependent hemodynamic changes (9). Importantly, these populations ofhumans all exhibit low levels of circulating renin (105). As low-reninhypertension accounts for a larger (27%) fraction of human essentialhypertensives than high-renin hypertension (16%) (51), it is unclearwhether therapeutic targeting of AVP may have been prematurelyoverlooked as an antihypertensive therapy for selected populations ofhypertensive patients.

Together, these findings have led us to question whether the elevationsin AVP are necessary to cause or maintain hypertension due tochronically elevated brain RAS activity and to probe the mechanism(s) ofaction of AVP in this context. We hypothesized that transgenicactivation of the brain RAS would elevate plasma AVP, and that actionsof AVP are required to induce hypertension by the brain RAS through somecombination of vasoconstriction and altered renal function. To examinethese hypotheses, we utilized a unique transgenic animal modelpreviously developed in our laboratory (29, 76). This double-transgenicmodel (the “sRA” model) takes advantage of the species specificity ofthe renin-mediated cleavage of angiotensinogen to cause brain-specifichyperactivity of the RAS. We have previously demonstrated that theseanimals exhibit a robust chronic hypertension, polydipsia, polyuria, andan elevated resting metabolic rate. Importantly, we have also previouslydetermined that sRA mice exhibit elevated plasma AVP levels and asuppression of the circulating RAS despite elevated renal sympatheticnerve activity (29). Here we demonstrate elevated neuronal AVPimmunostaining (specifically in the supraoptic nucleus), increased dailysecretion of AVP, robust desensitization of the vasculature of sRA miceto AVP, and the necessity of V2 AVP receptor signaling in themaintenance of hypertension and hyponatremia in this model. Thesefindings highlight a major role for AVP in the hypertension of sRA mice.

Materials and Methods:

Animals. All animal work was approved by the University of Iowa AnimalCare and Use Committee and was performed in accordance with the NationalInstitutes of Health “Guide for the Care and Use of Laboratory Animals.”

Double-transgenic (sRA) mice were generated as previously described (29,76). Briefly, “sR” mice expressing human renin under transcriptionalcontrol by the neuron-specific synapsin promoter were bred with “A” miceexpressing human angiotensinogen under transcriptional control by itsown promoter (line 11110/2×4284/1). Because of the species specificityof the reaction, human angiotensinogen is only cleaved to formangiotensin I by human renin. Hyperactivity of the RAS is therebyrestricted to sites of overlapping transgene expression in sRA offspring(i.e., subsections of the central nervous system that normally produceangiotensinogen).

Immunohistochemistry Immunohistochemical detection of AVP in the brainwas performed on 50 μm thick sections using a rabbit polyclonal antibodyto a synthetic peptide corresponding to the first six amino acids ofarginine8-vasopressin (Phoenix Pharmaceuticals, Burlingame, Calif.).Sections were cut from six (3 sRA, 3 wild type) brains perfusion fixedwith 4% paraformaldehyde and 0.5% glutaraldehyde and incubated in a1:1,000 dilution of antibody for 24 h at 4° C. The brains of sRA micewere “notched” for identification and incubated with sections fromwild-type animals. After incubation in a biotinylated goat anti-rabbitsecondary antibody and avidin-horseradish peroxidase, immunoreactivitywas detected using 3,3=-diaminobenzidine as a chromagen. On foursections from each animal, matched for rostrocaudal level,AVP-immunostained fragments larger than 10 μm were counted in the PVNand SON using ImageJ software from the NIH.

Blood pressure (tail-cuff). Here we first examined blood pressure in sRAmice using a Visitech Systems BP-2000 tail-cuff blood pressuremonitoring system, as previously described (84). Briefly, animals wereacclimated to warmed restraint boxes daily for 1 wk. Once acclimated, 30measurements of systolic blood pressure were averaged from each animaldaily for 2 wk to assess baseline blood pressure. Conivaptan (Vaprisol,YM 087, 22 ng/h sc, Baxter Healthcare) or tolvaptan (OPC-41061, 22 ng/hsc, Sigma Aldrich) was delivered to distinct subsets of mice by osmoticminipump (model 1004, Alzet). After osmotic minipump implantation,pressures were recorded daily for 10 days to assess drug effects.

Blood pressure (telemetry). Radiotelemetric blood pressures wererecorded from the carotid artery essentially as previously reported(29). Briefly, a telemeter probe (DSI, model TA11PA-C10) was insertedinto the common carotid artery under ketamine-xylazine anesthesia.After >2 days of recovery, blood pressure, heart rate, and spontaneousphysical activity were recorded for 30 s every 5 min using the Dataquestprogram (DSI). After baseline recordings, mice were chronicallydelivered conivaptan and tolvaptan via osmotic minipump that wasimplanted through an interscapular incision into the subcutaneous spaceof the back under isoflurane anesthesia.

Aortic Vascular Reactivity: Abdominal aortic rings were assessed forvascular reactivity as previously described (32). Briefly, mice wereeuthanized by overdose of pentobarbital (50 mg, i.p.), and the abdominalaorta was quickly removed and placed in Kreb's buffer containing (inmmol/L): 118.3 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 2.5CaCl2 and 11 glucose. Vascular rings (4-5 mm in length) were suspendedin oxygenated Kreb's buffer (95% O2/5% CO2) in organ baths at 37° C. andconnected to a force transducer via steel hooks. Resting tension wasadjusted to 0.5 grams over 45 minutes. Contractile responses were testedin response to AVP (10⁻¹⁰-10⁻⁶ mol/L), phenylephrine (PE, 10⁻⁸-3×10⁻⁵),endothelin-1 (ET-1, 10⁻¹⁰-10⁻⁷), prostaglandin F2α (PGF2α, 10⁻⁷-10⁻⁴),and angiotensin II (Ang II, 10⁻¹⁰-10⁻⁷). Following sub-maximalcontraction with PGF2α (40-50% of max; 3×10⁻⁶-6×10⁻⁶), relaxationresponses to acetylcholine (10⁻⁸-3×10⁻⁵) and sodium nitroprusside(10⁻⁹-10⁻⁵) were determined.

Mesenteric Artery Vascular Reactivity: Secondary branches of mesentericartery were dissected and placed in chilled oxygenated (21% O2, 5% CO2,and 74% N2) Kreb's buffer. A segment (˜1 mm long) of artery wastransferred to a vessel chamber (DMT), cannulated with glassmicropipettes and secured with silk ligatures. The artery was slowlypressurized to 40 mmHg without flow. After 30 min equilibration, vesselviability was tested by constriction response to 100 mM KCl. Vascularresponses to PE (10-9-10-5 mol/L), AVP (10-12-10-7 mol/L), and ET-1(10-11-10-8 mol/L) were then assessed. The artery was then superfusedwith calcium-free Krebs buffer containing 10-5 mol/L sodiumnitroprusside and 2 mmol/L EGTA to maximally dilate the vessel. Internaland external diameters were measured at 75 mm Hg. Wall thickness,media/lumen ratio and cross sectional area (CSA) were calculated aspreviously described by Neves, et al. (62).

Gene Expression: Mesenteric arteries (superior mesenteric arteryexcluded) and kidneys were snap frozen in liquid nitrogen and RNA wasextracted in Trizol®. Total RNA was isolated using an RNA Purelink®Minikit (Invitrogen) following the manufacturer's protocol.Concentrations were determined using a NanoDrop ND-1000. cDNA wasgenerated by RT-PCR using SuperScript III® (Invitrogen). qRT-PCR wasperformed using TaqMan gene expression assays (Applied Biosystems): RGS2(Mm00501385_m1), RGS5 (Mm00501393_m1), V1A (Mm00444092_m1), ETA(Mm01243722_m1), GAPDH (4352932E), or SYBR188 green assays (primersequences in Table 1: NKCC2, NCC, NHE3, ENaC-α, ENaC-β, ENaC-γ, NKA-α,V2R, AQP1, AQP2, AQP3, AQP4, PGES, and UT1-A) normalized againstβ-actin. SYBR-green reagents from Bio-Rad were utilized, and all realtime reactions were performed on a Bio-Rad iQ5 iCycle®.

TABLE 1 SYBR Green primer sequences for quantitative PCR. GenePrimer Sequences NKCC2 Forward: 5′-CCATGGTAACCTCTATCACTGGGT-3′SEQ ID NO. 1 Reverse: 5′-TCAAGCCTATTGACCCACCGAACT-3′ SEQ ID NO. 2 NCCForward: 5′-AAGTCGGGTGGCACCTATTTCCTT-3′ SEQ ID NO. 3Reverse: 5′-TTACGGTTTCTGCAAAGCCCACAG-3′ SEQ ID NO. 4 NHE3Forward: 5′-TCCTCTCAGCCATTGAGGACATCT-3′ SEQ ID NO. 5Reverse: 5′-ACTTTGCTGAGGAACTTCCGGTCA-3′ SEQ ID NO. 6 ENαCαForward: 5′-ACAATGGTTTGTCCCTGACACTGC-3′ SEQ ID NO. 7Reverse: 5′-TCACGTTGAAGCCACCATCATCCA-3′ SEQ ID NO. 8 ENαCβForward: 5′-TCTGCCAACCCTGGGACTGAATTT-3′ SEQ ID NO. 9Reverse: 5′-TGGCATAGATGCCCTCCTCTCTAA-3′ SEQ ID NO. 10 ENαCγForward: 5′-GCCAATCAGTGTGCAAGCAATCCT-3′ SEQ ID NO. 11Reverse: 5′-TTATTTGCTGGCTTTGGTCCCAGG-3′ SEQ ID NO. 12 Nα-K ATPase-αForward: 5′-TGAAGCTGACACCACGGAGAATCA-3′ SEQ ID NO. 13Reverse: 5′-TGCCGCTTAAGAATAGGCAGGTT-3′ SEQ ID NO. 14 V2RForward: 5′-TGTGATTGTCTACGTGCTGTGCTG-3′ SEQ ID NO. 15Reverse: 5′-GGGTTGGTACAGCTGTTAAGGCTA-3′ SEQ ID NO. 16 AQP1Forward: 5′-CTGGGCATTGAGATCATTGGCACT-3′ SEQ ID NO. 17Reverse: 5′-TGATACCGCAGCCAGTGTAGTCAA-3′ SEQ ID NO. 18 AQP2Forward: 5′-TAGCCCTGCTCTCTCCATTGGTTT-3′ SEQ ID NO. 19Reverse: 5′-AAACTTGCCAGTGACAACTGCTGG-3′ SEQ ID NO. 20 AQP3Forward: 5′-ATGGTGGCTTCCTCACCATCAACT-3′ SEQ ID NO. 21Reverse: 5′-AGGAAGCACATTGCGAAGGTCACA-3′ SEQ ID NO. 22 AQP4Forward: 5′-TGCCAGCTGTGATTCCAAACGAAC-3′ SEQ ID NO. 23Reverse: 5′-TCCCATGATAACTGCGGGTCCAAA-3′ SEQ ID NO. 24 PGESForward: 5′-TTTGCAACAAGTACTGGCCCATGC-3′ SEQ ID NO. 25Reverse: 5′-TGTTCGGTACACGTTGGGAGAGAT-3′ SEQ ID NO. 26 UT1-AForward: 5′-CACTGGCGACATGAAGGAATGCAA-3′ SEQ ID NO. 27Reverse: 5′-GGGTTGTTGACAAACATCACCTGAGC-3′ SEQ ID NO. 28 B-actinForward 5′-CATCCTCTTCCTCCCTGGAGAAGA-3′ SEQ ID NO. 29Reverse 5′-ACAGGATTCCATACCCAAGAAGGAAGG-3′ SEQ ID NO. 30

Blood and urine analyses: Plasma was obtained by collecting whole bloodby submandibular bleed into lithium heparin coated tubes, thencentrifuged at 5,000×g for 5 minutes, and the supernatant transferred toa fresh tube and frozen at −80° C. until analysis. Urine was collectedusing Nalgene single-mouse metabolism cages as previously described(29). Copeptin was measured using an ELISA kit (USCN Life Sciences),according to the manufacturer's instructions. Blood chemistries andurine creatinine were determined using a handheld iSTAT clinicalchemistry analyzer (Abbott), with CHEM8+ cartridges. Urine protein wasdetermined using a bicinchoninic acid assay kit (Thermo Fisher/Pierce),according to the manufacturer's instructions.

Statistics: Data were analyzed by ANOVA with repeated measures asappropriate. Post-hoc analyses were performed using Bonferronimultiple-comparisons procedures. EC50 and maximum response calculationswere performed by fitting individual dose-response data sets to afour-parameter logistic function (Hillslope method); y=min+(max204min)/(1+(x/EC50)̂Hillslope). All mRNA fold changes were calculated usingthe Livak method (54). All analytical comparisons were performed usingSigmaStat/SigmaPlot (Systat). All data are presented as mean±sem.

Results:

In both sRA and wild-type animals, AVP immunoreactivity was observed inthe cells in the suprachiasmatic (SCN), SON, PVN, and circular nuclei ofthe hypothalamus as expected (41, 100). AVP-immunoreactive fibers weretraceable from the SON and PVN to the median eminence (FIG. 2A). Thoughthere was no obvious difference in the numbers of AVP immunoreactiveneurons in the SCN and PVN between sRA and wild-type animals, neuronaland fiber immunoreactivity was consistently denser in the sRA animals.The most striking difference between sRA and control animals was thedoubling of the number of AVP immunoreactive neurons detected in theretrochiasmatic part of the SON in sRA animals (FIGS. 2A and B) comparedwith the retrochiasmatic SON in wild-type animals. Copeptin is theCOOH-terminal fragment of the fully translated AVP proprotein and istherefore translated in a 1:1 molar ratio with AVP. Because it exhibitsa far greater biological half-life than AVP, it has been proposed as amore reliable measure of chronic AVP release than AVP itself (90).Copeptin levels were significantly reduced in plasma from sRA mice (FIG.2C). Because of its small size (38 amino acids, 4.22 kDa), however, thisprotein is rapidly cleared from the plasma by the kidneys. Copeptinconcentrations appeared elevated in the urine from sRA mice, though thedifference was not significantly different. After we accounted for thegrossly elevated (˜7-fold) urine production rate of sRA mice, however,it is clear that the total daily copeptin clearance into urine isgrossly elevated in sRA mice (˜20-fold, FIG. 2D). These data togetherindicate that there is an approximate 20-fold increase in AVP secretionin sRA mice. This large difference in total daily copeptin loss to urinewas still present (8-fold) after normalization for total daily urinecreatinine (creatinine: control, 0.20±0.03 vs. sRA, 0.40±0.05 mg/day,P<0.01, and copeptin/creatinine: control, 49±36 vs. sRA, 406±176 pg/mg,P=0.05) or (10-fold) after normalization total daily urine protein(protein: control, 41±4 vs. sRA, 86±9 mg/day, P<0.01, andcopeptin/protein: control, 180±100 vs. sRA, 1,845±665 mg/mg, P=0.02).

Under baseline conditions, sRA mice exhibited a hypertension that waseasily detectable by tail-cuff (FIG. 3A). These data replicate ourpreviously published measures of hypertension in this model, asdetermined by direct cannulas and by radiotelemetry (29, 76). Chronicsubcutaneous infusion of the nonselective, nonpeptide AVP V1A/V2receptor antagonist conivaptan resulted in a complete normalization ofthe hypertension in sRA mice. Continuous recording of blood pressures inan exemplar sRA mouse at baseline and during 18 days of continuoussubcutaneous conivaptan infusion documented a gradual but substantialreduction in blood pressure (FIG. 3B) that was paralleled by a slightreduction in heart rate (FIG. 3C). Importantly, spontaneous physicalactivity remained normal throughout the recording period, suggestingthat the animal was not lethargic or otherwise ill due to the surgeryand conivaptan infusion (FIG. 3D).

To dissect the relative contributions of various vasopressin receptorsubtypes in the hypertension of sRA mice, we next examined the bloodpressure consequences of chronic subcutaneous infusion of theV2-selective antagonist tolvaptan. Chronic infusion of tolvaptan causeda nearly identical normalization of blood pressure (FIG. 3E) to thatobserved with conivaptan (FIG. 3A). Importantly, this blood pressurereduction was confirmed in a cohort of sRA mice tested usingradiotelemetry (FIG. 3F).

Additional evidence for chronic hypertension and vasopressin-specificchanges in sRA mice comes from vascular reactivity assays. First,abdominal aortic rings were examined ex vivo for reactivity to selectedvasoconstrictor and vasodilator compounds. Aortic rings from sRA miceexhibited normal constrictor responses to potassium chloride (FIG. 4A).Abdominal aortic rings exhibited a robust rightward shift in responsesto the vasodilator acetylcholine (Table 2), but normal responses tosodium nitroprusside, indicating endothelial dysfunction typical inchronic hypertension models (FIGS. 4, B and C). Supporting a chronicelevation in AVP levels, abdominal aortas from sRA mice exhibited arobust suppression of constrictor responses to AVP (FIG. 4D), reflectedboth in a trend toward a rightward (reduced) potency shift and asignificant suppression of maximal response (Table 2). No potency orefficacy changes were observed in contractile responses to PE, ET-1, ANGII, or PGF2α (FIG. 4, E-H), suggesting AVP specific changes in the sRAvasculature.

TABLE 2 Potency and efficacy analyses of various vasoactive compounds inabdominal aortas and 2°-branch mesenteric artery of male sRA and controllittermate mice. EC₅₀ Maximum Response Compound Control sRA Control sRAAbdominal aorta nmol/l nmol/l g g Phenylephrine 1.900 ± 250 2.430 ± 6201.18 ± 0.05 1.02 ± 0.10 Angiotensin II  2.26 ± 0.45  4.24 ± 1.14 0.32 ±0.03 0.38 ± 0.03 Arginine vasopressin  2.19 ± 0.19  3.72 ± 0.73* 0.29 ±0.03 0.12 ± 0.01† Prostaglandin F_(2α) 4.600 ± 750 3.600 ± 290 1.59 ±0.06 1.50 ± 0.10 Endothelin-1  3.40 ± 0.33  5.19 ± 0.93 0.45 ± 0.04 0.55± 0.09 nmol/l nmol/l % % Acetylcholine   130 ± 35   879 ± 298* 78.9 ±3.3 70.4 ± 8.9 Sodium nitroprusside  29.4 ± 3.7  34.2 ± 1.7 91.5 ± 0.593.5 ± 0.8 Mesenteric artery nmol/l nmol/l % % Phenylephrine 1.460 ± 660  560 ± 80 67.3 ± 3.0 64.5 ± 4.1 Arginine vasopressin  0.57 ± 0.15 15.89± 13.46 52.5 ± 3.2 17.3 ± 6.5† Endothelin-1  1.68 ± 0.43  0.45 ± 0.1767.8 ± 4.3 58.0 ± 4.6 Data are presented as means ± SE. Aortas: Control,n = 6; sRA, n = 5. Mesenteric artery: Control, n = 6; sRA, n = 6. *P ≤0.05, and †P ≤ 0.001 vs. Control.

Acknowledging that smaller arteries are important in controllingperipheral resistance, vascular reactivity of second order branches ofmesenteric arteries were next examined using pressurized myography.Mesenteric artery branches exhibited a significant reduction incontractile response to potassium chloride (FIG. 5A); however,normalization of other constrictor responses to this lower KCl responsein sRA mice had no qualitative effect on data interpretation (notshown). Similar to abdominal aortic rings, mesenteric arteries from sRAmice exhibited a trend toward a rightward shift and a substantialsuppression of maximal response (Table 2) to AVP (FIG. 5B).

Mesenteric arteries exhibited normal contractile responses to PE, withno change in efficacy or potency. In response to ET-1, mesentericarteries from sRA mice exhibited a normal maximal response and a smallbut statistically significant leftward potency shift. These data confirman AVP-specific desensitization in smaller arteries of sRA mice, furthersupporting the conclusion that AVP is chronically elevated in sRA mice.Mesenteric arteries from sRA mice exhibited substantial eutrophic inwardremodeling, providing further evidence of chronic hypertension in thismodel. While no difference in external diameter was detected betweencontrol and sRA mice (FIG. 5C), lumen diameter was significantly smallerin sRA mice because of increased wall thickness. This resulted in anincreased media-tolumen ratio but no significant change incross-sectional area.

To explain the reduced vascular reactivity to AVP, we next measuredexpression of the V1A receptor. Mesenteric arteries from sRA miceexhibited significantly suppressed V1A receptor mRNA but no change inETA receptor expression (FIG. 5D). Furthermore, there was a selectivedownregulation of regulator of G protein signaling-2 (RGS2) expressionbut no change in RGS5 expression.

In contrast to vascular V1A downregulation, renal V2 receptors andaquaporin-2 mRNA levels were unchanged in sRA mice (Table 3). The onlyrenal transporter that showed significant changes in expression in sRAmice was the sodium chloride cotransporter (NCC, 5-fold of control,P<0.05), though the sodium/hydrogen exchanger (NHE) showed a trendtoward reduction (NHE3, 0.6-fold of control, P=0.08) and the ENaC-αsubunit showed a trend toward elevation (ENaC-α, 10-fold of control,P=0.08). It should be noted that these renal gene expression assays wereperformed on only male sRA and littermate control mice, and thestatistical power is low due to a small number of replicates per group(n=4 each). Thus it is possible that the changes in NHE3 and ENaC-α mayboth be physiologically significant.

TABLE 3 Renal expression of selected receptors and transporters in sRAand littermate control mice. t-test Gene Control (n = 4) sRA (n = 4) PValue AVPR2 1.000 (0.840-1.191)  0.778 (0.583-1.038) 0.425 NCC 1.000(0.736-1.359)  5.232 (3.850-7.112) 0.009 NHE3 1.000 (0.825-1.212)  0.605(0.522-0.701) 0.075 NKCC2 1.000 (0.632-1.581)  0.621 (0.415-0.929) 0.464ENaCα 1.000 (0.468-2.135) 10.021 (4.556-22.041) 0.080 ENacβ 1.000(0.657-1.522)  0.596 (0.282-1.263) 0.611 ENacγ 1.000 (0.669-1.495) 1.682 (1.120-2.525) 0.398 Na-K-ATPase-α 1.000 (0.620-1.613)  0.819(0.553-1.215) 0.744 AQP1 1.000 (0.610-1.640)  0.643 (0.511-0.808) 0.441AQP2 1.000 (0.747-1.338)  0.541 (0.440-0.665) 0.133 AQP3 1.000(0.636-1.571)  0.317 (0.192-0.524) 0.640 AQP4 1.000 (0.397-2.521)  0.483(0.237-0.983) 0.506 PGES 1.000 (0.779-1.283)  0.616 (0.237-1.597) 0.164UT1-A 1.000 (0.486-2.057)  0.569 (0.406-0.799) 0.643 Data presented asfold-of-control: means ± SE. See text for abbreviations and moreinformation.

Finally, to more directly probe a V2-mediated mechanism in thecardiovascular phenotypes of sRA mice, we examined blood chemistryresponses to tolvaptan (Table 4). We previously documented anapproximate 4 mM hyponatremia in sRA mice under baseline conditions(29). Here we determined that sRA mice were hyponatremic (FIG. 6A) andhypocalcemic (FIG. 6B), and chronic tolvaptan delivery corrected both ofthese imbalances (genotype X drug interaction P<0.05 for both). sRA micealso exhibited alterations in chloride, total CO2, glucose, blood ureanitrogen, creatinine, hematocrit, and anion gap, and whereas tolvaptantreatment did affect some of these endpoints (potassium, chloride, andblood urea nitrogen), it did so in a manner independent of genotype asno genotype X drug interactions were uncovered (Table 4).

TABLE 4 Blood chemistry at baseline or following tolvaptan infusion insRA and control littermate mice. Females Males Baseline TolvaptanBaseline Tolvaptan Control sRA Control sRA Control sRA Control sRAParameter (n = 12) (n = 8) (n = 5) (n = 6) (n = 8) (n = 3) (n = 4) (n =4) Females Age, wk 23.5 ± 0.1 23.6 ± 0.2 22.7 ± 0.2 22.6 ± 0.2 19.4 ±1.1 18.5 ± 1.3 22.5 ± 0.3 22.5 ± 0.3 Sodium, mM^(G, T, G×T) 147.3 ± 0.7 142.6 ± 1.2  146.2 ± 0.7  146.3 ± 0.7  147.4 ± 0.9  141.4 ± 2.8  148.3 ±1.3  147.5 ± 1.9  Potassium, mM^(S)  6.6 ± 0.3  6.5 ± 0.5  6.4 ± 0.1 5.3 ± 0.3  6.5 ± 0.5  6.8 ± 0.7  6.6 ± 0.5  5.4 ± 0.1 Chloride,mM^(G, S, T) 115.7 ± 1.0  110.3 ± 1.7  112.4 ± 0.5  105.2 ± 1.6  119.4 ±2.0  115.8 ± 2.2  115.5 ± 0.3  107.0 ± 2.7  Ionized calcium,  1.15 ±0.04  1.01 ± 0.04  1.19 ± 0.05  1.18 ± 0.03  1.07 ± 0.06  0.78 ± 0.12 1.18 ± 0.04  1.18 ± 0.03 mM^(G, T, G×T) Total CO₂, mM^(G, T) 18.4 ± 1.121.6 ± 2.1 24.0 ± 1.1 28.8 ± 1.7 17.0 ± 1.2 18.8 ± 1.7 24.0 ± 0.8 26.0 ±3.4 Glucose, mg/dl^(G, S×T) 215 ± 8  193 ± 16 190 ± 13 136 ± 15 196 ± 11155 ± 16 182 ± 7  181 ± 28 Bun, mg/dl^(G, T) 22.4 ± 1.5 42.4 ± 4.9 18.4± 0.9 24.5 ± 1.7 28.5 ± 4.0 40.8 ± 9.0 21.5 ± 0.9 27.3 ± 2.1 Creatinine,mg/dl^(G*)  0.21 ± 0.01  0.33 ± 0.06  0.24 ± 0.02  0.33 ± 0.04  0.21 ±0.01  0.24 ± 0.02  0.25 ± 0.03  0.30 ± 0.04 Hematocrit, % RBC^(G, G×S)45.8 ± 0.5 52.6 ± 0.6 45.4 ± 0.7 49.8 ± 1.1 43.8 ± 0.7 52.6 ± 0.9 43.8 ±1.1 52.0 ± 1.1 Anion gap, mM^(G, G×S) 21.2 ± 0.6 18.4 ± 1.0 17.0 ± 1.518.5 ± 0.7 18.3 ± 1.4 14.8 ± 4.4 16.3 ± 0.5 21.0 ± 2.4 Values are means± SE. Three-way ANOVA results: ^(G)P < 0.05 main effect of genotype,^(S)P < 0.05 main effect of sex, ^(T)P < 0.05 main effect of tolvaptan(22 ag/h, 10 days sc), ^(G×S)P < 0.05 genotype × sex interaction,^(G×T)P < 0.05 genotype/tolvaptan interaction, ^(S×T)P < 0.05 sex ×tolvaptan interaction, *Lower detection limit for creatinine assay was0.30 mg/dl; values below detection were assigned value of 0.20. All endpoints were evaluated from check capillary blood collected inlithium-heparin coated tubes and tested using CHEMS+ cartridges in aniSTAT handheld chemistry analyzer (Abbot Labs).

Discussion

Here we examined a unique double-transgenic mouse model to test thehypothesis that AVP is required for the hypertension induced by thebrain RAS. Immunohistochemical examination of the brain revealedelevated AVP levels in the retrochiasmatic part of the supraoptichypothalamic nucleus but no consistent change in PVN immunostaining insRA mice. Confirming a required role for AVP signaling in thehypertension, chronic blockade of vasopressin V1A/V2 receptors resultedin normalization of blood pressure in sRA mice. While vascularreactivity in multiple arteries to PE, ET-1, ANG II, and PGF2α werelargely unchanged in sRA mice, responses to AVP were greatlydesensitized. Selective inhibition of V2 receptors had a potentantihypertensive action in sRA mice and normalized the hyponatremiatypical of this model. Together, these data strongly support a requiredrole for AVP, acting at V2 receptors, in the maintenance of brainRAS-derived hypertension.

Increased AVP signaling has been suggested as a mechanism for thehypertension in many models. Mice with either tightly regulated orstrongly overexpressed transgenic hyperactivity of the RAS throughoutthe body require elevated AVP signaling to maintain hypertension (19,61). Deoxycorticosterone acetate (DOCA)-salt hypertension, which isdependent on elevated brain RAS activity (40, 50, 69), also depends onAVP signaling. DOCA-salt treatment results in elevated plasma AVP levels(16, 57, 60, 99). Intracerebroventricular infusion of theangiotensin-converting enzyme inhibitor captopril into rats bothprevented and reversed DOCA-salt hypertension and was associated with areduction in plasma vasopressin levels despite a reduced blood pressure(40). The dependence of DOCA-salt hypertension on AVP has also beendemonstrated using AVP-deficient Brattleboro rats, as the hypertensiveeffects of DOCA-salt are greatly diminished in these animals (16, 106).Complimenting these findings from various hypertensive models,TGR(ASrAOGEN) rats, which exhibit reduced glial production ofangiotensinogen, are hypotensive and have reduced plasma AVP levels(79). These animals also exhibit altered patterns of AVP V1A receptorexpression within the brain (11), further supporting a brain RAS-AVPinteraction. Mice deficient for the V1A AVP receptor are hypotensive,though the relative importance of brain, vascular, cardiac, thrombocyte,and hepatic receptors is unclear (2, 48).

Effects of the RAS on the production and release of AVP were reported asearly as 1970, when Bonjour and Melvin (6) demonstrated thatperipherally administered renin or angiotensin II resulted indose-dependent increases in plasma AVP in dogs. Evidence for directactions of angiotensin on AVP release within the brain was provided byex vivo experiments using isolated rat neurohypophysis (23).Electrolytic lesion of the subfornical organ (39) or transection ofefferent projections from the subfornical organ (47) both attenuate therelease of AVP into the plasma in response to intravenous ANG II. Thusthe demonstrations here of elevated brain AVP staining and increaseddaily copeptin (and thereby AVP) release in sRA transgenic mice wereexpected. Further work is required to causally link specific RASreceptor subtypes to the AVP elevation, as morphological and functionalevidence support roles for both AT1 and AT2 receptors in AVP release.

The strongly increased AVP immunoreactivity in the SON implicates ANGII-mediated hyperactivity in the supraopticneurohypophysial pathway asleading to elevated AVP in sRA mice. ANG II injections into the SONdepolarize neurosecretory cells (100), ANG II-immunoreactive neurons andaxon terminals are found in the rodent SON intermingled with AVPimmunoreactive neurons, and ANG II and AVP are colocalized in someneurons (41). It is thus likely that local production and/or actions ofANG II within the SON regulate AVP production and secretion.

AVP is an endogenous agonist for at least four subtypes of receptors.The V1A receptor subtype is primarily found in the vasculature, signalsprimarily through Gaq, and mediates vasoconstriction. V1A receptors arealso present in neurons and appear to signal through cAMP to regulateneuronal function (2, 96). The V1B receptor subtype is primarily foundin the brain, signals through Gaq, and stimulates adrenocorticotropichormone. The V2 receptor subtype is primarily found in the collectingduct of kidney nephrons, signals through Gαs, and stimulates waterreabsorption through aquaporin mobilization. There is some evidence forexpression of V2 receptors in extrarenal tissues such as lung (22) andcerebellum (45), though their physiological significance in thesetissues is unclear. Finally, AVP is also an agonist at the VACM-1receptor, also known as Cullin-5, where it elicits calcium mobilizationin endothelial cells and renal collecting ducts (7, 8). Ourdetermination that mesenteric artery V1A receptors were downregulated insRA mice but renal V2 receptor expression was unchanged may suggest agreater role for AVP-mediated renal water retention in the hypertensionof sRA mice. Though not directly tested herein, this conclusion issupported by the slow time course for the effects of conivaptan (severaldays of infusion to see an effect, FIG. 3B), the antihypertensiveeffects of tolvaptan (FIGS. 3, E and F), and the normalization ofbaseline hyponatremia and hypocalcemia in this model (Table 4 and FIG.6) that are typical of the syndrome of inappropriate secretion ofantidiuretic hormone (SIADH) (25). RGS2 is expressed throughout thecardiovascular system and acts to negatively regulate Gaq-mediated GPCRsignaling, and therefore oppose vasoconstrictor responses (80). Studiesin human patients have revealed a negative correlation between RGS2expression and blood pressure, with hypertensive patients showingreduced RGS2 expression and hypotensive patients exhibiting elevatedRGS2 expression (37, 80, 101). A similar correlation is observed inhypertensive animal models (10, 11) and was again observed in thepresent study (FIG. 5D). RGS2 is known to be regulated in atissue-specific manner, and within cardiovascular tissues RGS2 iscontrolled through multiple biphasic mechanisms (104). Acute activationof Gaq by various hormone/receptor combinations upregulates RGS2rapidly, possibly to serve as a negative feedback mechanism. Incontrast, chronic stimulation of Gaq systems appears to cause a tonicsuppression of RGS2 expression (10, 11, 95, 104). Mice deficient forRGS2 exhibit robust hypertension due to chronic increases in peripheralvasoconstriction (33, 36). Vasopressin-induced calcium transients invascular smooth muscle cells from RGS2 knockout mice are augmented,highlighting the relationship between RGS2 and AVP signaling, presumablythrough V1A receptors (92) as these receptors utilize Gaq signaling (2,96). RGS2 knockout mice also exhibit substantially greater end-organdamage from chronic hypertension than do wild-type animals (87). RGS2also attenuates cAMP signaling in the kidney through modulation ofadenylyl cyclases (31, 91), which may result in modulation of AVPsignaling through V2 receptors, Gαs, and cAMP. Indeed, modulation ofRGS2 greatly affects renal V2 receptor signaling and the renal effectsof AVP in vivo (77). Thus it is tempting to speculate that modulation ofRGS2 in various tissues, along with elevated AVP signaling, maycontribute to the maintenance of hypertension in the context ofchronically elevated brain RAS activity. Differential regulationpatterns for V1A receptors and V2 receptors in pathological states havepreviously been described. Gózdz et al. (28) previously demonstratedthat in the TGR(mRen2)27 rat model of high-renin hypertension, cardiacV1A receptors are upregulated compared with control Sprague-Dawley rats,while renal V2 receptors are unchanged between strains. Trinder et al.(94) previously demonstrated that in the streptozotocin-injection modelof Type 1 diabetes mellitus, rats exhibited reduced hepatic and renalexpression of V1 receptors and AVP-induced inositol phosphateproduction, while renal V2 receptors and AVP-induced cAMP production areagain unchanged. Thus our observation that vascular V1A receptors weredownregulated and vascular reactivity to AVP was desensitized whilerenal V2 receptors and their function were largely unchanged is notunprecedented.

Previously, we demonstrated a robust (twofold) elevation in plasma AVPlevels in female sRA mice under baseline conditions (collected 4 h intothe light phase of a 12:12 light-dark cycle), and this difference wasnot detected in males (29). The doubling of plasma AVP concentration wasachieved in sRA males as well, following a very brief (4 h) waterrestriction that had no effect on plasma AVP in control males. In thepresent study we determined that copeptin loss to urine (the majormechanism for clearance of this 4-kDa peptide) was the same in both maleand female mice (FIG. 2D). While urine copeptin measures relate to therate of AVP secretion, direct plasma AVP measures relate to both AVPsecretion and degradation/clearance. Thus we now hypothesize that AVPsecretion rates are similarly elevated in both male and female sRA mice,but that there exist sex-specific differences in the rates of AVPdegradation/clearance. The determination that AVP receptor blockadeeffectively eliminated hypertension in both sexes in the present study(FIG. 3) further supports this hypothesis. Studies into the sex-specificdifferences in AVP clearance mechanisms are ongoing.

Perspectives and Significance: Collectively, our data support a model ofelevated brain RAS activity driving an increase in AVP secretion. AVPaction upon V2 receptors subsequently contributes to elevated bloodpressure and hyponatremia. We hypothesize that these effects aremediated through excessive water retention, which when combined with theextreme polydipsia of this model, results in a polyuria phenotypepossibly through a pressure-diuresis mechanism. Based on the well-knownfunction of V2 receptors in renal collecting duct aquaporin-2mobilization, we suspect a renal-mediated mechanism is hyperactive insRA mice, though we have not here directly examined the localization ofthe V2 receptors responsible for the observed antihypertensive actionsof tolvaptan. These data may support the use of the sRA mouse as anexperimental model of the SIADH (25) or other diseases characterized byelevated AVP production or reduced clearance. The brain-specificgeneration and action of angiotensin peptides is gaining substantialinterest for the regulation of cardiovascular function, fluid balance,metabolic control, and even learning and memory. Vasopressin is alsowell-recognized for its role in fluid balance, blood pressureregulation, and various behaviors (pair bonding, altruism, learning,memory, fluid, and food intake), and its production and release are wellknown to be stimulated by angiotensins within the brain. Therefore, theimplication of vasopressin as a primary mediator of angiotensinergichypertension simultaneously 1) identifies vasopressin as a possiblemediator of other newly recognized functions of the brain RAS (e.g.,metabolic control, learning and memory, etc.); and 2) identifiesangiotensinsensitive, vasopressin-producing brain structures (e.g., thesupraoptic nucleus) as major cardiovascular regulatory centers that maydeserve substantially more investigation for therapeutically targetinghypertension and other disorders, especially in selected humanpopulations with low-renin hypertension (3, 4, 9, 21, 26, 51, 66, 67,105).

Example 2. Early First Trimester Prediction of Preeclampsia by Copeptin:is Vasopressin Hypersecretion an Initiating Event in the Pathogenesis ofPreeclampsia?

Preeclampsia affects 5-7% of all pregnancies, approximately 300,000 peryear in the U.S. Yet, it disproportionately causes 15% of allmaternal-fetal morbidity and mortality (78). Preeclampsia is known tocause immediate and long term maternal-fetal morbidities such as fetalgrowth restriction, maternal-fetal death, and future adult neurologicaland cardiovascular diseases for mother and child (24, 42, 55, 56, 97,98). Because its pathogenesis is poorly understood, preventative,therapeutic, and curative modalities for preeclampsia are elusive. Thisemphasizes the importance of finding appropriate unifying pathways to beable to predict and treat preeclampsia. One potential pathway is thevasopressin pathway.

Vasopressin exhibits a short biological half-life (on the order of 5-20minutes in blood), which complicates direct measurement of this hormone.Vasopressin is translated in 1:1 stoichiometric ratio with a small,inactive pro-segment, copeptin. Copeptin is eliminated primarily byrenal excretion and is very stable in plasma. Consequently, it is a veryuseful biomarker for vasopressin secretion (3). Zulfikaroglu et al.(108) recently documented a late second/early third trimester elevationin circulating copeptin in preeclamptics. Furthermore, selectedpopulations exhibit vasopressin-dependent hypertension, includingAfrican Americans, the elderly, and patients in chronic heart or renalfailure (3, 4, 9, 21). These populations are also characterized by lowcirculating renin-angiotensin system activity. Interestingly, relativeto normotensive pregnancies, preeclamptic pregnancies also exhibitreduced circulating activity of the renin-angiotensin system (35). Thesedata lead us to hypothesize a potential causative role for vasopressinhypersecretion in the development of preeclampsia, and the possibleutility of copeptin as a novel predictive biomarker for preeclampsia inearly pregnancy.

Methods

Biosample and Clinical Data Acquisition: Maternal plasma and clinicalpatient information were obtained through the University of IowaIRB-approved (IRB#200910784) Maternal Fetal Tissue Bank (MFTB). In thisbank, pregnant women are prospectively recruited from the beginning oftheir prenatal care. MFTB inclusion criteria include any women >18 yearsold receiving prenatal care at the University of Iowa Hospitals &Clinics who are English speaking. The MFTB exclusion criteria includeprisoners, HIV+ or Hepatitis C positive women. Women who enroll into theMFTB provide a maternal blood sample for research whenever they haveclinically indicated blood draws throughout pregnancy. All maternalblood in the MFTB is uniformly processed. Maternal plasma and the buffycoat are isolated, aliquoted, and stored at −80° C. Maternal andneonatal clinical data obtained by the MFTB is obtained via dataextraction from the electronic medical record using standardized dataextraction forms. Extracted clinical data is routinely monitored foraccuracy and completeness by two of the authors (MKS and DAS).Additional data is also extracted by bioinformatics collaborators fromthe University of Iowa Institute for Clinical and Translational Sciencewho are able to query the central electronic medical record database.

Cohort Assembly: Inclusion criteria for preeclampsia cases includedwomen who delivered at UIHC, were enrolled in the MFTB, and carried thediagnosis of preeclampsia at the time of delivery. The diagnosis andclassification of cases of preeclampsia were based on the standardAmerican College of Obstetrics and Gynecology (ACOG) definitions foranalysis (1). These cases were identified by cross-referencing the MFTBdatabase with the bioinformatics query of mild and severe preeclampsiaICD-9 codes (642.4x, 642.5x, 642.7x, 642.9x) of bank participants at thetime of delivery. The electronic medical record of each potential casewas evaluated to confirm the diagnosis of preeclampsia by the ACOGdefinitions. Maternal age-matched plasma samples and correspondingclinical data for the control population were obtained by utilizing theMFTB database. The gestational age at the time of the collection of thesamples were classified by trimesters: first trimester (<13 completedgestational weeks), second trimester (13-26 completed gestationalweeks), and third trimester (>26 weeks).

Procedures: All maternal plasma copeptin concentrations were measured induplicate using a commercial enzyme-linked immunosorbent assay (ELISA)specific for human copeptin (USCN Life Science, Inc, Houston, Tex.). Theassay was performed according to the manufacturer's instructions. Theminimum detectible dose of human copeptin for this assay was 5.4 pg/mL.The intra-assay coefficient of variation is <10% and the interassaycoefficient of variation is <12%. To examine if renal function orvasopressin degradation throughout pregnancy affected copeptinconcentration, plasma Cystatin C (Sigma-Aldrich, St. Louis, Mo.) andvasopressinase (LNPEP, USCN Life Science, Inc, Houston, Tex.) weremeasured in duplicate in all samples utilizing commercial ELISA kits.

Animal Studies: Female C57Bl/6J mice were obtained from JacksonLaboratories between 8-12 weeks of age. Osmotic minipumps infusingvasopressin (240 ng/hr) or saline vehicle were inserted into thesubcutaneous space via incision between the scapulae. Following threedays of recovery, females were mated with male C57Bl/6J mice. Presenceof a vaginal plug indicated gestational day 0.5. Blood pressure wastracked before mating and throughout gestation by tail-cuffplethysmography. On gestational day 18, females were sacrificed fornecropsy. Pup weight was recorded. Dam kidney sections were generatedand imaged by electron microscopy by the University of Iowa Departmentof Pathology. All studies were approved by the University of Iowa AnimalCare and Use Committee (ACURF#1211239).

Statistical Analyses: The major aim of this study was to determine ifdifferences in first-trimester copeptin concentrations between pregnantwomen who did and did not develop preeclampsia predicted the developmentof preeclampsia. Using the smallest effect size in late gestationmaternal plasma copeptin concentrations from Zulfikaroglu et al. betweencontrol (310 pg/mL) and mild preeclamptics (620 pg/mL) with the largestreported standard deviation of 180 pg/mL, power of 80% and α=0.05, only7 participants per group are required. In order to account for aparsimonious, mixed effects regression model of 3 variables, a minimumof 30 samples per group was utilized.

All statistical analyses were performed with SigmaPlot 12.0 software(Systat Software, Inc, California) and confirmed using SAS 9.1 software(SAS Institute Inc, Cary, N.C.). Stepwise regression was used to developa model for this dataset and to evaluate for possible confounding.Logistic regression models were constructed and receiver operatingcharacteristic curves were constructed for regression diagnostics. Inaddition, chi square or Fisher exact test was utilized for categoricalvariables. For continuous variables, the Student's t-test or if criteriafor normality were not met, Mann-Whitney test was utilized. Allvariables were tested at significance level of 0.05.

Results

A total of 30 individual control (C) subjects and 51 individualpreeclamptic (P) subjects were utilized in this study. A full complementof first (C=12, P=20), second (C=10, P=20), and third (C=30, P=51),trimester plasma samples were not available for each participant.Maternal age, gravida, body mass index, percentage of those with chronichypertension and preexisting diabetes were similar between the controland preeclamptic groups (Table 5). In addition, the racial distributionbetween these groups were also similar and reflective of the Iowapopulation with a predominantly Caucasian populace based on current Iowacensus data. Of these maternal characteristics, only history ofpreeclampsia was significantly higher in the control group vs. thepreeclamptic group (53.3% vs. 17.7%, p=0.002). When evaluating thedelivery characteristics between the two groups (Table 5), typicaldifferences were observed between groups. The preeclampsia groupexhibited a significantly lower gestational age at delivery (36.2 vs.38.7 weeks, P=0.001), higher percentage of twin gestation (21.6 vs. 0%,P=0.016), and lower birthweight (2777.0 vs. 3424.0 grams, P=0.0001).These findings are consistent with the known morbidities associated withpreeclampsia: higher rate of preterm delivery, higher rate of twingestation, and lower birthweight due to vascular causes and earlierdelivery (85).

TABLE 5 Group Characteristics. Non- Pre- pregnant Control eclampsiaGroup Characteristics (n = 33) (n = 31) (n = 50) P Value MaternalCharacteristics Maternal Age (years) 31.4 30.0 30.0 0.86 Gravida 1.3 2.62.7 <0.001 Body Mass Index (kg/m²) 29.6 30.4 31.9 0.48 Chronic Essential 9.1% 29.0% 20.0% 0.13 (χ² = 4.1) Hypertension Preexisting Diabetes 3.0%  9.7% 10.0% 0.47 (χ² = 1.5) History of Preeclampsia   0% 51.6%18.0% 0.002 (χ² = 25.7) Race: Caucasian, 90.9% 87.1% 90.0% 0.63 (χ² =6.2) not Hispanic Race: Hispanic   0%  6.5%  4.0% 0.63 (χ² = 6.2) Race:Asian  6.1%  3.2%   0% 0.63 (χ² = 6.2) Race: African-American  3.0% 3.2%  2.1% 0.63 (χ² = 6.2) Pregnancy Characteristics Gestational Age38.7 36.2 0.001 at Delivery (wk) Mode of Delivery: 53.3% 34.0% 0.09 (χ²= 4.75) Vaginal Mode of Delivery: 40.0% 64.0% 0.09 (χ² = 4.75) C-SectionMode of Delivery:  6.7%  2.0% 0.09 (χ² = 4.75) Operative VaginalDelivery Twin Gestation   0% 21.6% 0.016 (χ² = 5.76) Birthweight (grams)3424.0 2777.0 <0.001 1 minute APGAR 7.2 7.3 0.95 5 minute APGAR 8.7 8.50.49

As seen in FIG. 7A, measurement of the maternal plasma copeptinconcentration revealed a significant increase in mean copeptin inpregnant women who developed preeclampsia in comparison with control,non-preeclamptic women in the first trimester (2045 vs. 903 pg/mL,p=0.008), second trimester (1806 vs. 706 pg/mL, p=0.001), and thirdtrimester (1890 vs. 822 pg/mL, p=0.0006). These group differences inplasma copeptin are likely not associated with changes in renal functionand vasopressin degradation as measured by plasma Cystatin C andVasopressinase respectively as these levels were similar between groupsin each trimester (FIGS. 7B and 7C).

Given this significant increase in copeptin, we constructed receiveroperating characteristic curves for each trimester to interrogate ifmaternal plasma copeptinconcentration was predictive of the developmentof preeclampsia. Furthermore, optimal copeptin concentration cutoffswere determined from these curves. As seen in FIG. 8, the ROCsdemonstrated significant areas under the curve in the first trimester(AUC=0.80, p=0.005), second trimester (AUC=0.87, p=0.002), and thirdtrimester (AUC=0.72, p=0.004). These data indicate that the meanmaternal plasma copeptin concentration is predictive of the developmentof preeclampsia.

Further, we determined if clinically significant covariates would alterthe association of the development of preeclampsia and copeptinconcentration at particular trimesters. Logistic regression models wereconstructed with the diagnosis of preeclampsia as the dependentvariable. Participants were dichotomized according to being above orbelow the determined cutoff for a particular trimester. Using thetrimester specific cutoff values (first trimester: 1018 pg/mL, secondtrimester: 943 pg/mL, third trimester: 860 pg/mL), models were generatedusing the status of being above or below the cutoff as an independentvariable while controlling for clinically significant covariates. Aftercontrolling for clinically significant covariates such as maternal age,body mass index, diabetes, chronic hypertension, history ofpreeclampsia, and twin gestation, copeptin concentration was stillsignificantly associated with the development of preeclampsia in thefirst, second and third trimester (Table 6). With the exception of themodel including the second trimester [copeptin] cutoff and a history ofpreeclampsia, all models significantly predict the development ofpreeclampsia. These results confirm our observation that copeptinconcentration is significantly elevated in the plasma of pregnant womenwho will develop preeclampsia in comparison to controls. This robustelevation in copeptin concentration occurs early in the first trimesterand remains elevated throughout pregnancy despite potential confoundingeffects of clinically significant obstetrical and vascular covariates.Finally, we observed that the chronic elevation of vasopressin duringpregnancy is sufficient to cause preeclampsia-like phenotypes in mice.Vasopressin infusion significantly reduced the rate of pregnancy (FIG.9A), highlighting a role for this hormone in reproductivepathophysiology. Vasopressin infusion during successful pregnancyresulted in cardinal preeclampsia phenotypes, including a robustincrease in blood pressure and apparent proteinuria (FIG. 9B),substantial fetal growth restriction (FIG. 9C) and pathognomicglomerular endotheliosis (FIG. 9D).

TABLE 6 Using first, second and third trimester specific cutoffs,maternal plasma copeptin remains significantly predictive of thedevelopment of preeclampsia despite adjustment of significant clinicalcovariates. First Trimester Model [Copeptin] Beta Adjusted Odds P Cutoff= 1018 pg/mL [Copeptin] Ratio Value 1st Trimester [Copeptin] 1.8 6.050.025 1st Trimester [Copeptin] + 2.2 9.03 0.018 Maternal Age 1stTrimester [Copeptin] + 1.8 6.05 0.026 Body Mass Index 1st Trimester 2.18.17 0.024 [Copeptin] + Diabetes 1st Trimester [Copeptin] + 1.9 6.690.024 Chronic Essential Hypertension 1st Trimester [Copeptin] + 2.613.46 0.028 History of Preeclampsia 1st Trimester [Copeptin] + 1.6 4.950.05 Twin Gestation Second Trimester Model Beta Adjusted Odds P[Copeptin] Cutoff = 943 pg/mL [Copeptin] Ratio Value 2nd Trimester[Copeptin] 2.8 16.44 <0.001 2nd Trimester [Copeptin] + 3.1 22.20 <0.001Maternal Age 2nd Trimester [Copeptin] + 2.9 18.17 <0.001 Body Mass Index2nd Trimester 2.8 16.44 <0.001 [Copeptin] + Diabetes 2nd Trimester[Copeptin] + 3.2 24.53 <0.001 Chronic Essential Hypertension 2ndTrimester [Copeptin] + 20 485165195.41 0.995 History of Preeclampsia 2ndTrimester [Copeptin] + 3.1 22.20 0.0047 Twin Gestation Third TrimesterModel Beta Adjusted Odds P [Copeptin] Cutoff = 860 pg/mL [Copeptin]Ratio Value 3rd Trimester [Copeptin] 1.3 3.67 0.017 3rd Trimester[Copeptin] + 1.3 3.67 0.017 Maternal Age 3rd Trimester [Copeptin] + 1.44.06 0.012 Body Mass Index 3rd Trimester 1.7 5.47 0.008 [Copeptin] +Diabetes 3rd Trimester [Copeptin] + 1.3 3.67 0.017 Chronic EssentialHypertension 3rd Trimester [Copeptin] + 1.3 3.67 0.038 History ofPreeclampsia 3rd Trimester [Copeptin] + 1.6 4.95 0.008 Twin Gestation

Our data demonstrates that copeptin is a strong predictor of thedevelopment of preeclampsia. More importantly, it is predictive of thedevelopment of preeclampsia throughout pregnancy as early as the sixthgestational week. This finding represents a major advance in theprediction of preeclampsia. Currently, anti-angiogenic factors likesFLT-1 and Endoglin are elevated as early as 12 weeks before thediagnosis of preeclampsia (52). Follow up analyses of sFLT-1, Endoglin,and other anti-angiogenic factors suggest that testing characteristicsof these factors are poor in application to clinical practice (46).Furthermore, a limitation of these factors is that the significantchanges in antiangiogenic factors overall have been reported to occuronly as early as the second trimester.

In recent years, substantial effort has been invested to identify firsttrimester predictors of preeclampsia. These investigations have includedfirst trimester circulating hyperglycosylated human chorionicgonadotropin (hCG) (43), Interleukin-1β (83), high sensitivityC-reactive protein (44), and Pregnancy-associated plasma protein-A(PAPPA) (17). These factors have been shown to be poor to moderatelypredictive of preeclampsia. Given the promise of antiangiogenic markersin the pathogenesis of preeclampsia, they have been investigated in thefirst trimester. In conjunction with uterine artery Doppler (UAD)analyses, these factors have been shown to only be moderately predictive(AUC=0.74) of preeclampsia (64). An elevated uterine artery Dopplerpulsatile index in the first trimester is correlated with thedevelopment of preeclampsia. Poon et al. demonstrated that UAD coupledwith maternal history and aneuploidy markers in the first trimester canbe very predictive of preeclampsia with AUC=0.96. In and of itself, UADshave an AUC=0.91 (71, 72). Although this may be a powerful tool,reliable UAD requires substantial training for sonographers to decreasesignificant interassay variability through verified programs such as theFetal Medicine Foundation (68). Such training may not be as available inall hospital settings. Clearly, there is utility in finding a simplepredictor of preeclampsia as early in pregnancy as possible, andcopeptin represents the first simple and individually predictivebiomarker. Coupling plasma copeptin measures with other knownfirst-trimester assays may further increase predictive power. Multipleprocesses involving placental dysregulation, endothelial celldysfunction, immunology, oxidative stress, altered vascular biology, andangiogenesis make finding a singular cause of preeclampsia nearlyimpossible. As preeclampsia is a disease resulting from multiplepathways, the development of a predictive model and the search for atherapeutic pathway for preeclampsia treatment may need to come from theupstream regulators or inducers of these multiple pathways. Vasopressinsits at the crux of many of these pathways. The acknowledgement ofcopeptin, and thereby vasopressin secretion, as a novel, veryearly-pregnancy diagnostic biomarker for preeclampsia, plus results fromour vasopressin-infused mouse model collectively support the hypothesisthat elevated vasopressin secretion in early pregnancy may contribute tothe development of preeclampsia. Arginine vasopressin is a peptidehormone synthesized primarily within magnocellular neurons of thesupraoptic nucleus and paraventricular nuclei of the brain, though it isproduced by selected peripheral tissues in small quantities. Axonalprojections from magnocellular neurons comprise the posterior pituitarygland, and upon stimulation vasopressin is released into thecirculation. Vasopressin then acts upon multiple receptor types toultimately increase blood volume, vascular constriction, and reduceosmolality (75).

The connection of vasopressin to the pathogenesis of preeclampsia isstrengthened by the immunoactive nature of vasopressin and theimmunologic initiating events of preeclampsia. As reviewed by Russelland Walley (75), and by Chikanza and Grossman (12), vasopressin has avariety of immunomodulatory effects. Depending on site of action anddose, vasopressin is known to affect and be affected by tumor necrosisfactor-α, interleukin-1β, interferon-γ, β-endorphin, and prostaglandinE2—many of which are altered in preeclampsia. Vasopressin is known tostimulate the autologous mixed lymphocyte response. Vasopressin isproduced by, and acts upon, human T cells, B cells, andmonocytes/macrophages. High doses of vasopressin cause an amplificationof prostaglandin E2 synthesis by human dermal fibroblasts. Further,vasopressin-deficient hypertension Brattleboro rats exhibit substantialchanges in circulating immune cell populations and function, includingincreased neutrophils. These data suggest a potential link between theelevated vasopressin secretion in early pregnancy observed in thepresent study with excessive peripheral immune activation, and thesubsequent development of preeclampsia. Based on our data and others, wetherefore posit that vasopressin may play an important role ininitiating the immunologic milieu that precipitates preeclampsia.

Our study has benefitted from the high quality of clinical data andbiosample fidelity provided by the Maternal Fetal Tissue Bank.Furthermore, our study was strengthened by being appropriately poweredto evaluate our desired outcomes. One weakness of our study is thepredominantly Caucasian population in Iowa. Even though the relationshipof copeptin and preeclampsia is robust after clinical covariateadjustment, we are not appropriately powered to analyze the variance dueto race. A larger sample size would be necessary for that analysisdespite finding significant covariate adjusted associations.

The temporal organization of molecular events and clinical associationsthat define preeclampsia has been somewhat muddled to date, asessentially all known mechanisms occur or develop in rapid successionduring late-pregnancy. Our data clearly demonstrate an early-pregnancyelevation in vasopressin secretion, thus aligning all other knownmechanisms as potential targets of vasopressin action. These resultshighlight the utility of plasma vasopressin/copeptin measurements in theprediction of preeclampsia, and are consistent with a potentialcausative role for vasopressin in preeclampsia. While our data from micedemonstrate the sufficiency of vasopressin to cause preeclampsia-likephenotypes, future studies are required to elucidate the tissues,receptors, and mechanisms that mediate the induction of preeclampsia byvasopressin. Substantial clinical studies are required to assess thenecessity of vasopressin signaling for the development of preeclampsia,and the utility of targeting this system to treat the disorder. Finally,additional investigations will be required to identify the mechanismsthat induce excessive vasopressin secretion, to better understand theevent(s) that initiate preeclampsia.

Example 3. Vasopressin Infusion Causes Dose-Dependent Increase inLate-Pregnancy Blood Pressure

Applicants envisioned that chronic infusion of vasopressin duringpregnancy will increase blood pressure, as experienced by subjectssuffering from preeclampsia.

Method: Wildtype C57Bl/6J female mice were chronically infused withvaried doses of arginine vasopressin (Sigma-Aldrich), via subcutaneousosmotic minipumps (Alzet), for three days preceding mating, then throughgestational day 16. Mice were first acclimated to the restraint devicesused for recordings for one week (“week −3”) before data recordingsbegan. Blood pressure was assessed daily for two weeks precedingminipump implantation, through gestational day 16. Blood pressure wasmeasured using tail-cuff plethysmography (Visitech). On any givenrecording day, the mouse was restrained and lightly warmed throughoutthe (30 min) recording period. Thirty consecutive 1 mininflation/deflation cycles were performed and successful pressuredeterminations were averaged within recording period for each mouse.Data sets from individual days with fewer than 10 successfuldeterminations were excluded from analyses. Daily blood pressure valueswere averaged within animal for each week for statistical comparisons.

Results: Blood pressure was indistinguishable among groups for the twoweeks preceding pump implantation and mating, and again for the firsttwo weeks of pregnancy (FIG. 10). During the third week of pregnancy(gestational days 14-16, “week 3”), mice infused with vasopressin at 24ng/hr, s.c. demonstrated a significantly (P=0.03 by Tukeymultiple-comparisons procedure) increased blood pressure compared topregnant mice infused with only 2.4 ng/hr, s.c. vasopressin.

Discussion: Pregnancy is typically accompanied by a slow reduction, thennormalization of blood pressure. In contrast, preeclamptic pregnanciesexhibit relatively normal patterns of blood pressure control in earlytrimesters, which are then followed by a rapid onset of hypertension inthe third trimester. The data presented here illustrate the sufficiencyof vasopressin infusion (in a dose- and time-dependent manner) toincrease blood pressure during pregnancy. Notably, this increase inpressure occurs specifically in late pregnancy, which closely matchesthe time course of preeclampsia progression in humans. These databolster the assertion that vasopressin infusion represents the first(and currently, the only) animal model of the early-pregnancy eventsthat lead to preeclampsia, and that vasopressin hypersecretion/infusionis capable of inducing preeclampsia.

These conclusions are consistent with the concept that inhibitingvasopressin production/release/steady-state/action may represent a noveltherapeutic route to prevent and treat preeclampsia and/or reduce theseverity of the disease.

Example 4. Vasopressin Infusion During Pregnancy in Mice Results inSubstantial Intrauterine Growth Restriction and Spontaneous FetalResorption

Chronic infusion of vasopressin at 24 ng/hr, s.c. throughout pregnancy(3 days preceding through GD18) causes interrupted or restrictedfetoplacental development, a hallmark phenotype of preeclampticpregnancies.

Methods: Nine adult wildtype female mice were obtained from in-housebreeding colonies that are based on the C57Bl/6 background strain. Threedays before breeding, females were implanted with subcutaneous osmoticminipumps (Alzet) to continuously deliver arginine vasopressin (AVP,Sigma-Aldrich) at 24 ng/hr. Females were mated for two consecutivenights with wildtype male C57BL/6J males purchased from JacksonLaboratories. GD1 was defined based upon the presence of a vaginal spermplug. On gestational day 18, female mice were sacrificed by CO2asphyxiation, and fetuses and fetoplacental units were isolated by bluntdissection.

Results: Six of the nine female mice became pregnant despite the limitedmating time-course. Of these six dams, one (200372-3) carried sevenfetuses of relatively normal size compared to fetuses of historical mice(0.90-0.99 grams/fetus for C57BL/6 mice, and 0.97-1.00 grams/fetus forDBA/1 mice, (10)). Regardless, as a group, fetuses across all sixpregnancies were substantially smaller than historical controls (10)(0.4929±0.0397 gram/fetus, n=48, P<0.0001 by one-sample t-test against0.90 gram historical control C57Bl/6 fetus mass; summarized in Table 7).Three dams carried a mixture of growth-restricted fetuses plusfetoplacental units that had necrotized and begun the resorption process(see FIG. 11 for examples), bringing the total number of resorbedfetoplacental units to 17%.

Discussion: Intrauterine growth restriction and fetal resorption areunusual events in unchallenged wildtype mice. Gendron demonstrated thatroughly 2% of fetoplacental units undergo resorption in CFW/SW×DBA/2crosses (27). Sulila reported a resorption frequency of between 4-7% forC57 mice and 0-13% for DBA mice (86). Mattsson reported a resorptionfrequency of between 3-7% for C57 females mated to CBA males (58). Inthe current experiment, 17% of fetoplacental units were resorbed. Fiveof six pregnant mice exhibited abnormal pregnancies involving growthrestriction and/or fetal resorption (e.g.—in the two pregnanciesdepicted in FIG. 11, eight of fourteen fetoplacental units wereresorbed, and all eight remaining fetuses were growth-restricted), farbeyond the expected rate of such events. These data highlight thepowerfully negative effects that elevated vasopressin can have on thefetoplacental unit during pregnancy, and are therefore consistent withour assertion that vasopressin is sufficient to cause key phenotypes ofpreeclampsia such as growth restriction or death.

Example 5. Chronic Infusion of Vasopressin During Pregnancy in C57BL/6JFemale Mice Results in Dose-Dependent Proteinuria and IntrauterineGrowth Restriction

Hypothesis: Chronic infusion of vasopressin during pregnancy will induceproteinuria and intrauterine growth restriction, hallmark phenotypes ofpreeclampsia.

Method: Eighteen female C57BL/6J mice were obtained from the JacksonLaboratories. Subcutaneous osmotic minipumps (Alzet) were implanted todeliver vasopressin at 0.24, 2.4, or 24 ng/hr (six mice each group).After three days, mice were mated with wildtype C57BL/6J male mice forone night (thereby defining gestational day zero). On GD16, mice wereplaced into single-mouse metabolic cages (Nalgene) to collect urine fortwo consecutive nights. On GD18, mice were sacrificed to quantify andcollect fetuses and placentas. Urine protein was assessed bycommercially-available BCA assay (Pierce).

Results: Of the six mice infused with each dose of vasopressin, threebecame pregnant during 24 ng/hr infusion; five became pregnant during2.4 ng/hr infusion; and two became pregnant during 0.24 ng/hr infusion.In pregnant mice, urine protein content was elevated in a dose-dependentmanner with vasopressin infusion (FIG. 12). Similar effects wereobserved in non-pregnant mice (not shown).

Fetal masses were significantly suppressed for dams infused with 24ng/hr vasopressin compared to 2.4 ng/hr vasopressin (FIG. 13). Placentalmasses were indistinguishable across treatment groups.

In the 24 ng/hr vasopressin infusion group, one pregnant mouse exhibitedpreterm labor (FIG. 14). This dam began delivering pups spontaneously ongestational day 17. Laboratory personnel intervened as quickly aspossible to sacrifice the dam, to collect placentas and fetuses and toquantify the frequency of resorbed fetoplacental units.

Discussion: Vasopressin infusion during pregnancy resulted in adose-dependent increase in severity of proteinuria. Further, the currentcohort of vasopressin-infused dams carried growth-restricted fetuses.Specifically, the highest vasopressin dose resulted in greater growthrestriction than the medium and lowest doses, but notably all threegroups exhibit substantially suppressed fetal masses compared tohistorical data for C57BL/6 mice (0.9-1.0 grams, (27, 58, 86)).

Preterm labor is an extremely rare event in non-human animals such asmice (74). Thus the induction of preterm labor in one of the high-dosevasopressin infused dams is very notable. Together, these dataillustrate that elevated vasopressin levels during pregnancy cause majorphenotypes of preeclampsia including proteinuria (and thereby kidneydamage), intrauterine growth restriction, and even preterm labor.

Example 6. Inhibition of Vasopressin Secretion by Tetrahydrobiopterin(BH4)

Summary: Inhibition of vasopressin production & secretion may representa novel therapeutic approach to prevent or treat preeclampsia. Weexamined the utility of daily BH4 treatment (once daily injection, 10mg/kg/day, i.p.) to inhibit vasopressin secretion in mice.

Background & Hypothesis: Double-transgenic “sRA” mice express humanrenin via the neuron-specific synapsin promoter, and humanangiotensinogen via its own promoter (29, 76). This results in chronicincreases in the generation of angiotensin peptides within the brain,which results in polydipsia, polyuria, and elevated resting metabolicrate. sRA mice also exhibit robust hypertension that is mediatedthrough, and dependent upon, gross elevations in vasopressin secretionrates (53). Notably sRA mice maintain a baseline hyponatremia,underscoring the concept that the source of stimulation of vasopressinhypersecretion in this model is independent of dehydration.

BH4 is used clinically to treat phenylketonuria (PKU), and is markedunder the names “Kuvan” and “Sapropterin.” BH4 has previously been shownto interfere with vasopressin regulation in the neurohypophysis. Inisolated neurointermediate lobes, BH4 reduces vasopressin content underbaseline and potassium-stimulated conditions. In rats, BH4 reducesneurohypophysial vasopressin under euhydrated but not dehydratedconditions (13, 14). Together these results suggest that BH4specifically modulates hypothalamic vasospressin production to selected(non-dehydration) stimuli.

As preeclampsia is characterized as a state of elevated vasopressinsecretion in the absence of dehydration stimuli, we propose the generalhypothesis that BH4 will reduce vasopressin secretion duringpreeclampsia. Specifically, here we hypothesized that BH4 treatmentwould reduce vasopressin levels in sRA mice.

Methods: Double-transgenic sRA mice and littermate controls were placedinto single-mouse metabolic cages (Nalgene), and maintained on a 12:12light:dark cycle at 22° C. with ad libitum access to standard chow(Teklad 7013) and water. Mice were injected once daily (i.p.) with BH4to achieve 10 mg/kg/day. On the fourth day, mice were sacrificed by CO2asphyxiation and trunk blood was collected into lithium heparin-coatedtubes and placed on ice between 11:30 AM and 1:00 PM (5.5 to 7 hoursinto the light phase of the standard light cycle). Blood was thencentrifuged at 2,000×g for 20 minutes. Plasma was collected and storedat −80° C. until analysis. Plasma samples were analyzed for vasopressinusing a commercially-available ELISA kit (Cayman, catalog #583951),according to the manufacturer's instructions. Results falling outsidethe range of the standard curve (>3,000 pg/mL or <23.4 pg/mL) were setto the maximum or minimum (3,000 or 23.4 pg/mL), respectively, forstatistical comparisons. Data were analyzed by three-way ANOVA followedby Tukey all-pairwise multiple comparison procedures.

Results: Plasma vasopressin values were indistinguishable inwater-replete control and littermate sRA mice treated with vehicle forthree days. This is similar to our previous demonstration thatsteady-state plasma levels of vasopressin are relatively normal or onlyslightly elevated in water-replete sRA mice, with females exhibiting asmall elevation and males exhibiting no change (29). Treatment ofcontrol mice with BH4 for three days had no effect on steady-state AVPlevels. In contrast, treatment of sRA mice with BH4 for three dayscaused a significant (P=0.017) reduction in plasma AVP levels (FIG. 15).

Conclusion: From these data we conclude that steady-state levels of AVPare significantly reduced in sRA mice following a relatively short-termtreatment with BH4. For this preliminary study we used the dose of BH4that is used for human patients suffering from phenylketonuria (10mg/kg/day), despite the elevated metabolism exhibited by mice. Further,the short timecourse of treatment (3 days) was selected based on thepharmacokinetics previously demonstrated for humans (3). Thus, it wouldbe reasonable to believe that similar or greater efficacy would beobserved in human patients.

Collectively, these data are consistent with the hypothesis thatelevated (dehydration-independent) vasopressin secretion can becountered by administration of BH4. These data support the hypothesisthat BH4 or related compounds represent a novel therapeutic interventionto suppress preeclampsia-related elevations in vasopressin secretion.Thus, in light of our demonstration that elevated vasopressin ispredictive of preeclampsia in humans and sufficient to phenocopypreeclampsia in mice, BH4 and related compounds delivered to a pregnantpatient may represent a novel therapeutic intervention to reduce theincidence and/or severity of preeclampsia.

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What is claimed is:
 1. A method of treating preeclampsia in a subject inneed thereof, comprising inhibiting production and/or secretion and oreffects of AVP or lowering the concentration of AVP in the blood of thesubject.
 2. The method of claim 1, wherein the method comprisesadministering to the subject a therapeutically effective amount of apharmaceutical compound that inhibits production and/or secretion and/oreffects of AVP in the subject.
 3. The method of claim 2, wherein thepharmaceutical compound inhibits the effects of AVP by inhibiting anarginine vasopressin receptor.
 4. The method of claim 3, wherein thearginine vasopressin receptor comprises at least one of V1A, V2, andV1B.
 5. The method of claim 2, wherein the pharmaceutical compound istetrahydrobiopterin (BH4) or a chemically related compound.
 6. Themethod of claim 2, wherein the pharmaceutical compound is a vasopressinreceptor antagonist.
 7. The method of claim 6, wherein thepharmaceutical compound is selected from the group consisting ofconivaptan, tolvaptan, and relcovaptan, and combinations thereof.
 8. Themethod of claim 1, wherein the concentration of AVP is lowered using anextracorporeal therapy technique.
 10. The method of claim 8, wherein theextracorporeal therapy technique comprises at least one of apheresis,hemodialysis, and hemofiltration.
 11. A composition for treatment ofpreeclampsia, comprising: a therapeutically effective amount of a firstarginine vasopressin receptor inhibitor; and a therapeutically effectiveamount of a second arginine vasopressin receptor inhibitor.
 12. Thecomposition of claim 11, wherein the first arginine vasopressin receptorinhibitor comprises a first vaptan drug and the second argininevasopressin receptor inhibitor comprises a second vaptan drug.
 13. Apharmaceutical dosage form comprising the composition of claim 12 andone or more pharmaceutically suitable carriers, diluent, and/orexcipients.
 14. The pharmaceutical dosage form of claim 13, wherein thedosage form comprises an oral, injection, infusion, inhalation,transdermal, or implant dosage form.
 15. A composition for treatment ofpreeclampsia, comprising a therapeutically effective amount oftetrahydrobiopterin (BH4) or a chemically related compound; and atherapeutically effective amount of an arginine vasopressin receptorinhibitor.